CHAPTER 5
MOLECULAR BIOCHEMICAL amp PHYSIOLOGICAL ANALYSIS OF
TRANSGENIC TOBACCO AND TOMATO PLANTS
50 ABSTRACT
The transgenic tobacco and tomato plants harbouring carrot AFP were analysed
The integration of AFP in tobacco and tomato plants was confirmed by Southern blot
All further analysis was carried out in T1 generation AFP expression level in plant
organs after cold stress was studied in tobacco by sq RT-PCR The expression analysis
revealed that AFP is stably expressed in all the transgenic lines of tobacco and tomato
The expression of AFP in vegetative and reproductive tissues was also confirmed
The transcript accumulation of various antioxidant enzymes and signaling molecules after
cold stress in transgenic tobacco plants revealed that the expression of all the genes were
high in WT plants during cold stress while the transgenic lines showed a steady
expression level Transgenic plants subjected to the chilling stress showed a significant
decrease in membrane injury index compared to the WT which was determined by
electrolytic leakage assay The growth of transgenic plants was normal as that of WT
tobacco plants in normal conditions but inhibited during cold stress
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51 INTRODUCTION
Plants have a multi-facetted response to low temperature stress and they combat
the stress by bringing a myriad of molecular biochemical cellular and physiological
changes (Singh et al 2002 Thomashow 1999) These changes are acquired by the
change in photosynthetic rates calcium influxes activation of kinasephosphatise
cascades (Singh et al 2002) alteration in the membrane lipid composition (Kung 1998)
accumulation of osmolytes like proline glycine betaine and soluble sugars (Chen amp
Murata 2002) and increasing the levels of various antioxidants (Prasad et al 1994)
A notable upregulation or down regulation in the specific gene transcripts of the above
mentioned process occurs when a low temperature stress is thrusted on the plants
With the onset of low temperature stress putative thermo sensors like Calcium
messengers at the cell membrane generates a series of stress signals that are further
transmitted and amplified through a cascade that include Ca2++ signaling and a stepwise
kinasephosphatase chain This cascade ultimately reaches the nucleus and transcription
factors which act as ―master switch to activate the gene expression Low temperature
activates transcription of many genes which are either up or downregulated (Shinozaki amp
Yamaguchi-Shinozaki 2000) The upregulated genes encodes mainly 2 groups of
proteins the first group includes proteins playing active role in stress tolerance LEA
proteins antifreeze proteins water channel proteins enzymes involved in sugar and
proline synthesis detoxification enzymes etc The second group includes those involved
in regulation of signal transduction and gene expression which are activated during the
stress that includes protein kinases transcription factors and the enzymes in phospholipid
metabolism like phospholipases This alteration in gene expression and accumulation of
202
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other organic molecules like osmolytes protects the cell from low temperature induced
damages However some plants adapt to the low temperature stress which is very
complex and the exact molecular biochemical and physiological changes are poorly
understood
512 Antioxidant Enzymes- It is widely reported that the injury to the plant is caused
by stress and it is related to oxidative damage at the cellular level In plants most of the
degenerative reactions are allied with various abiotic biotic stresses that are due to toxic
Reactive Oxygen Species (ROS) Free radical scavenging antioxidant enzymes either
catalyzes the reactions by quenching ROS (without being transformed into a destructive
radical itself) or process directly Although they are all often induced in similar stress
situations they mostly show differential expression in response to specific stress
(Adam et al 1995) Organelles like chloroplasts and mitochondria are major source of
ROS production in plant cells because of high oxidizing metabolic activity or due to the
high rate of electron flow In plants plastids are the main source of ROS which produces
high amounts of superoxide radicals and hydrogen peroxide especially during reduced rate
of carbon fixation which are common during abiotic stresses (Takahashi amp Murata 2005)
In addition to the superoxide and hydrogen peroxide chloroplasts can also produce
singlet oxygen through chlorophyll excitation after the absorption of light by the pigment
systems Mitochondria are the major source of ROS in non-photosynthetic tissues
(Navrot et al 2007) The third intracellular source of ROS is the peroxisomes They
contain many oxidases that produce H2O2 and O2-
as byproducts of the metabolic
reactions which they catalyze The glycolate oxidase is one of the important enzymes
located in peroxisome specifically relevant during abiotic stresses In addition to the
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above ROS sources in the presence of redox-active metals hydroxyl radicals (OHbull) can
be formed from H2O2 through the Fentonlsquos reaction or from H2O2 and O2- through the
Haber-Weiss reaction The OHbull radicals are highly reactive that can cause extensive
oxidative damage in the cell (Moller et al 2007) Apart from the general metabolic
reactions various biotic and abiotic stresses increase ROS formation in plants
An increase in the free radicals in the cells can initiate severe oxidation of various cellular
components and thereby changing the cells redox status (Mittler et al 2004) hence
continuous control of ROS is very essential under stress conditions (Meyer et al 2007)
There exists equilibrium between the generation of ROS and their scavenging by various
antioxidative enzymes (Foyer et al 2005) This equilibrium may be perturbed by
different biotic and abiotic stress factors and the disturbances in equilibrium that lead to
increased levels of ROS which can cause significant damage to cell membranes
The general reaction catalysed by various antioxidant enzymes is as follows
Enzyme Reaction catalyzed
SOD O2 -
+ O2 - + 2H
+ harr 2H2O2 + O2
CAT 2H2O2 harr O2 + 2H2O
GPX 2GSH + PUFA-OOH harr GSSG + PUFA + 2H2O
GST RX + GSH harr HX + R-S-GSH
APX AA + H2O2 harr DHA + 2H2O
GR NADPH + GSSG harr NADP+ + 2GSH
(R may be aliphatic aromatic or heterocyclic group Ali amp Alqurainy 2006)
A variety of defence mechanisms have been suggested on the basis of the
biochemical and physiological responses against cold injuries Acclimation to low
temperature may be related to an enhanced antioxidant system by the activity of different
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scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which
prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved
different mechanisms to cope with the low temperature related oxidative stress Low
temperature induced accumulation of glutathione (GSH) has been observed in Picea abies
and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was
also induced in response to low temperature in soybean summer squash and wheat under
experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above
responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-
soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase
(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and
glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in
detoxification of ROS (fig 51)
Plants with high level of antioxidants or antioxidant enzymes are reported to have a
relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp
Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase
(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976
Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen
(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that
are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively
less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate
peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to
ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the
monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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functions of tocopherol ascorbate and glutathione In Photoprotection
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Environment 1211-215
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United States of America 923903-3907
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29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
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33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
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34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
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of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
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Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
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America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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51 INTRODUCTION
Plants have a multi-facetted response to low temperature stress and they combat
the stress by bringing a myriad of molecular biochemical cellular and physiological
changes (Singh et al 2002 Thomashow 1999) These changes are acquired by the
change in photosynthetic rates calcium influxes activation of kinasephosphatise
cascades (Singh et al 2002) alteration in the membrane lipid composition (Kung 1998)
accumulation of osmolytes like proline glycine betaine and soluble sugars (Chen amp
Murata 2002) and increasing the levels of various antioxidants (Prasad et al 1994)
A notable upregulation or down regulation in the specific gene transcripts of the above
mentioned process occurs when a low temperature stress is thrusted on the plants
With the onset of low temperature stress putative thermo sensors like Calcium
messengers at the cell membrane generates a series of stress signals that are further
transmitted and amplified through a cascade that include Ca2++ signaling and a stepwise
kinasephosphatase chain This cascade ultimately reaches the nucleus and transcription
factors which act as ―master switch to activate the gene expression Low temperature
activates transcription of many genes which are either up or downregulated (Shinozaki amp
Yamaguchi-Shinozaki 2000) The upregulated genes encodes mainly 2 groups of
proteins the first group includes proteins playing active role in stress tolerance LEA
proteins antifreeze proteins water channel proteins enzymes involved in sugar and
proline synthesis detoxification enzymes etc The second group includes those involved
in regulation of signal transduction and gene expression which are activated during the
stress that includes protein kinases transcription factors and the enzymes in phospholipid
metabolism like phospholipases This alteration in gene expression and accumulation of
202
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other organic molecules like osmolytes protects the cell from low temperature induced
damages However some plants adapt to the low temperature stress which is very
complex and the exact molecular biochemical and physiological changes are poorly
understood
512 Antioxidant Enzymes- It is widely reported that the injury to the plant is caused
by stress and it is related to oxidative damage at the cellular level In plants most of the
degenerative reactions are allied with various abiotic biotic stresses that are due to toxic
Reactive Oxygen Species (ROS) Free radical scavenging antioxidant enzymes either
catalyzes the reactions by quenching ROS (without being transformed into a destructive
radical itself) or process directly Although they are all often induced in similar stress
situations they mostly show differential expression in response to specific stress
(Adam et al 1995) Organelles like chloroplasts and mitochondria are major source of
ROS production in plant cells because of high oxidizing metabolic activity or due to the
high rate of electron flow In plants plastids are the main source of ROS which produces
high amounts of superoxide radicals and hydrogen peroxide especially during reduced rate
of carbon fixation which are common during abiotic stresses (Takahashi amp Murata 2005)
In addition to the superoxide and hydrogen peroxide chloroplasts can also produce
singlet oxygen through chlorophyll excitation after the absorption of light by the pigment
systems Mitochondria are the major source of ROS in non-photosynthetic tissues
(Navrot et al 2007) The third intracellular source of ROS is the peroxisomes They
contain many oxidases that produce H2O2 and O2-
as byproducts of the metabolic
reactions which they catalyze The glycolate oxidase is one of the important enzymes
located in peroxisome specifically relevant during abiotic stresses In addition to the
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above ROS sources in the presence of redox-active metals hydroxyl radicals (OHbull) can
be formed from H2O2 through the Fentonlsquos reaction or from H2O2 and O2- through the
Haber-Weiss reaction The OHbull radicals are highly reactive that can cause extensive
oxidative damage in the cell (Moller et al 2007) Apart from the general metabolic
reactions various biotic and abiotic stresses increase ROS formation in plants
An increase in the free radicals in the cells can initiate severe oxidation of various cellular
components and thereby changing the cells redox status (Mittler et al 2004) hence
continuous control of ROS is very essential under stress conditions (Meyer et al 2007)
There exists equilibrium between the generation of ROS and their scavenging by various
antioxidative enzymes (Foyer et al 2005) This equilibrium may be perturbed by
different biotic and abiotic stress factors and the disturbances in equilibrium that lead to
increased levels of ROS which can cause significant damage to cell membranes
The general reaction catalysed by various antioxidant enzymes is as follows
Enzyme Reaction catalyzed
SOD O2 -
+ O2 - + 2H
+ harr 2H2O2 + O2
CAT 2H2O2 harr O2 + 2H2O
GPX 2GSH + PUFA-OOH harr GSSG + PUFA + 2H2O
GST RX + GSH harr HX + R-S-GSH
APX AA + H2O2 harr DHA + 2H2O
GR NADPH + GSSG harr NADP+ + 2GSH
(R may be aliphatic aromatic or heterocyclic group Ali amp Alqurainy 2006)
A variety of defence mechanisms have been suggested on the basis of the
biochemical and physiological responses against cold injuries Acclimation to low
temperature may be related to an enhanced antioxidant system by the activity of different
204
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scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which
prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved
different mechanisms to cope with the low temperature related oxidative stress Low
temperature induced accumulation of glutathione (GSH) has been observed in Picea abies
and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was
also induced in response to low temperature in soybean summer squash and wheat under
experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above
responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-
soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase
(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and
glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in
detoxification of ROS (fig 51)
Plants with high level of antioxidants or antioxidant enzymes are reported to have a
relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp
Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase
(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976
Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen
(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that
are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively
less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate
peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to
ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the
monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid
205
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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other organic molecules like osmolytes protects the cell from low temperature induced
damages However some plants adapt to the low temperature stress which is very
complex and the exact molecular biochemical and physiological changes are poorly
understood
512 Antioxidant Enzymes- It is widely reported that the injury to the plant is caused
by stress and it is related to oxidative damage at the cellular level In plants most of the
degenerative reactions are allied with various abiotic biotic stresses that are due to toxic
Reactive Oxygen Species (ROS) Free radical scavenging antioxidant enzymes either
catalyzes the reactions by quenching ROS (without being transformed into a destructive
radical itself) or process directly Although they are all often induced in similar stress
situations they mostly show differential expression in response to specific stress
(Adam et al 1995) Organelles like chloroplasts and mitochondria are major source of
ROS production in plant cells because of high oxidizing metabolic activity or due to the
high rate of electron flow In plants plastids are the main source of ROS which produces
high amounts of superoxide radicals and hydrogen peroxide especially during reduced rate
of carbon fixation which are common during abiotic stresses (Takahashi amp Murata 2005)
In addition to the superoxide and hydrogen peroxide chloroplasts can also produce
singlet oxygen through chlorophyll excitation after the absorption of light by the pigment
systems Mitochondria are the major source of ROS in non-photosynthetic tissues
(Navrot et al 2007) The third intracellular source of ROS is the peroxisomes They
contain many oxidases that produce H2O2 and O2-
as byproducts of the metabolic
reactions which they catalyze The glycolate oxidase is one of the important enzymes
located in peroxisome specifically relevant during abiotic stresses In addition to the
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above ROS sources in the presence of redox-active metals hydroxyl radicals (OHbull) can
be formed from H2O2 through the Fentonlsquos reaction or from H2O2 and O2- through the
Haber-Weiss reaction The OHbull radicals are highly reactive that can cause extensive
oxidative damage in the cell (Moller et al 2007) Apart from the general metabolic
reactions various biotic and abiotic stresses increase ROS formation in plants
An increase in the free radicals in the cells can initiate severe oxidation of various cellular
components and thereby changing the cells redox status (Mittler et al 2004) hence
continuous control of ROS is very essential under stress conditions (Meyer et al 2007)
There exists equilibrium between the generation of ROS and their scavenging by various
antioxidative enzymes (Foyer et al 2005) This equilibrium may be perturbed by
different biotic and abiotic stress factors and the disturbances in equilibrium that lead to
increased levels of ROS which can cause significant damage to cell membranes
The general reaction catalysed by various antioxidant enzymes is as follows
Enzyme Reaction catalyzed
SOD O2 -
+ O2 - + 2H
+ harr 2H2O2 + O2
CAT 2H2O2 harr O2 + 2H2O
GPX 2GSH + PUFA-OOH harr GSSG + PUFA + 2H2O
GST RX + GSH harr HX + R-S-GSH
APX AA + H2O2 harr DHA + 2H2O
GR NADPH + GSSG harr NADP+ + 2GSH
(R may be aliphatic aromatic or heterocyclic group Ali amp Alqurainy 2006)
A variety of defence mechanisms have been suggested on the basis of the
biochemical and physiological responses against cold injuries Acclimation to low
temperature may be related to an enhanced antioxidant system by the activity of different
204
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scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which
prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved
different mechanisms to cope with the low temperature related oxidative stress Low
temperature induced accumulation of glutathione (GSH) has been observed in Picea abies
and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was
also induced in response to low temperature in soybean summer squash and wheat under
experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above
responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-
soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase
(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and
glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in
detoxification of ROS (fig 51)
Plants with high level of antioxidants or antioxidant enzymes are reported to have a
relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp
Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase
(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976
Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen
(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that
are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively
less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate
peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to
ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the
monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid
205
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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above ROS sources in the presence of redox-active metals hydroxyl radicals (OHbull) can
be formed from H2O2 through the Fentonlsquos reaction or from H2O2 and O2- through the
Haber-Weiss reaction The OHbull radicals are highly reactive that can cause extensive
oxidative damage in the cell (Moller et al 2007) Apart from the general metabolic
reactions various biotic and abiotic stresses increase ROS formation in plants
An increase in the free radicals in the cells can initiate severe oxidation of various cellular
components and thereby changing the cells redox status (Mittler et al 2004) hence
continuous control of ROS is very essential under stress conditions (Meyer et al 2007)
There exists equilibrium between the generation of ROS and their scavenging by various
antioxidative enzymes (Foyer et al 2005) This equilibrium may be perturbed by
different biotic and abiotic stress factors and the disturbances in equilibrium that lead to
increased levels of ROS which can cause significant damage to cell membranes
The general reaction catalysed by various antioxidant enzymes is as follows
Enzyme Reaction catalyzed
SOD O2 -
+ O2 - + 2H
+ harr 2H2O2 + O2
CAT 2H2O2 harr O2 + 2H2O
GPX 2GSH + PUFA-OOH harr GSSG + PUFA + 2H2O
GST RX + GSH harr HX + R-S-GSH
APX AA + H2O2 harr DHA + 2H2O
GR NADPH + GSSG harr NADP+ + 2GSH
(R may be aliphatic aromatic or heterocyclic group Ali amp Alqurainy 2006)
A variety of defence mechanisms have been suggested on the basis of the
biochemical and physiological responses against cold injuries Acclimation to low
temperature may be related to an enhanced antioxidant system by the activity of different
204
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scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which
prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved
different mechanisms to cope with the low temperature related oxidative stress Low
temperature induced accumulation of glutathione (GSH) has been observed in Picea abies
and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was
also induced in response to low temperature in soybean summer squash and wheat under
experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above
responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-
soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase
(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and
glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in
detoxification of ROS (fig 51)
Plants with high level of antioxidants or antioxidant enzymes are reported to have a
relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp
Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase
(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976
Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen
(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that
are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively
less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate
peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to
ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the
monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid
205
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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scavenging enzymesby molecules like Tocopherols β-Carotenes Lycopene etc which
prevents the accumulation of ROS (Prasad 1996) Different plant species have evolved
different mechanisms to cope with the low temperature related oxidative stress Low
temperature induced accumulation of glutathione (GSH) has been observed in Picea abies
and Pinus strobes during winter (Esterbauer amp Grill 1978 Anderson et al 1992) GSH was
also induced in response to low temperature in soybean summer squash and wheat under
experimental conditions (Vierheller amp Smith 1990 Wang 1995) In addition to the above
responses cells also synthesize lipid soluble antioxidants (tocopherol and β-carotene) water-
soluble reductants (ascorbate and glutathione) and enzymes such as superoxide dismutase
(SOD) catalase (CAT) ascorbate peroxidase (APX) Glutathione peroxidase (GPX) and
glutathione reductase (GR) (Zhang et al 1995) These enzymes have important roles in
detoxification of ROS (fig 51)
Plants with high level of antioxidants or antioxidant enzymes are reported to have a
relatively more tolerance to the most abiotic stress (Harper amp Harvey 1978 Madamanchi amp
Alcher 1991) The dehydro ascorbate reductase (DHAR) along with glutathione reductase
(GR) removes hydrogen peroxide through Halliwell-Asada pathway (Foyer amp Halliwell 1976
Nakano amp Asada 1980) SOD molecules are generated by the reaction of activated oxygen
(O2-) at PSI via Mehler reaction which is rapidly dismutate to peroxide by SOD enzyme that
are coupled to the thylakoid membranes thereby converting a harmful radicals to a relatively
less harmful molecule Thus peroxide produced is effectively scavenged by ascorbate
peroxidase (APX) The monodehydroascorbate radicals thus generated by APX is reduced to
ascorbic acid via ferridoxin or stromal monodehydroascorbate reductase Alternatively the
monodehydroascorbate are disproportionate into ascorbic acid and dehydroascorbic acid
205
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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which in turn will be converted to ascorbic acid by DHAR using reduced glutathione as an
electron donor Subsequent regeneration of reduced glutathione by glutathione reductase and
NADPH were reported by Bowler et al (1992)
Figure 51- ROS Scavenging Pathways in Different Plant Organelles
(A) The water-water cycle (WWC) (B) The ascorbate-glutathione cycle (AGC)
(C) The glutathione peroxidase cycle (GPXC) (D) Catalase (CAT)
APX ascorbate peroxidase AsA ascorbate CAT catalase DHA dehydroascorbate
DHAR dehydroascorbate reductase Gox glycolate oxidase GPX glutathione peroxidase
GR glutathione reductase GSH glutathione GSSG oxidized glutathione
MDHA monodehydroascorbate MDHAR monodehydroascorbate reductase NADPHox
NADPH oxidase PSI photosystem I and SOD superoxide dismutase (Peroni et al 2007)
513 Osmolytes in Stress Tolerance- Accumulation of low molecular weight metabolites
which acts as osmolytes in response to osmotic stress such as cold is a common phenomenon
(Bieleski 1982 Yancey et al 1982 Ford 1984) These osmolytes accumulate in the cells
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
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4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
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6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
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7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
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tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
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Plant Biology 5250-257
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plants Plant Journal 4215-223
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12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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during stress without interfering in normal metabolic processes and their accumulation
confers stress tolerance (McCue amp Hanson 1990 Delauney amp Verma 1993) The main
osmolytes include various sugar alcohols (like sorbitol and mannitol) iminoacid (proline)
and methylated tertiary or quaternary ammonium compounds (Glycine betaine) They act as
an osmolyte facilitating water retention in cytoplasm and thus allowing ion sequestration in
the vacuole The over expression of mannitol in transgenic tobacco chloroplasts resulted in
an improved tolerance to oxidative stress (Shen et al 1997) Transgenic tomato ectopically
expressing bacterial mannitol-1-phosphate dehydrogenase gene (mtlD) confers abiotic stress
tolerance (Khare et al 2010) Proline is thought to form adducts with the hydroxyl radicals
and thereby it reduces the damage caused by these species (Floyd amp Nagy 1984) Proline
thus performs various functions in plants and also acts as a rescue molecule which is
accumulated under the adverse conditions
514 Signaling during Stress Responses- The mechanism by which plants perceive
various stress signals and transmit them to cellular machinery to activate stress responses
are necessary for developing strategies to improve stress tolerance A signal transduction
pathway is initiated with the perception of stress signal followed by the production of
second messengers such as inositol phosphates and ROS The drought cold and salinity
stresses have been reported to induce a transient calcium influx into the cytoplasm
(Sanders et al 1999 Knight 2000) Ligand sensitive calcium channels control this internal
calcium ion release and the importance is the presence of repetitive transient bursts The first
round of transient calcium generation leads to generation of secondary signaling molecules
like ABA and ROS which further stimulate calcium release These multiple rounds of bursts
in calcium signaling from different sources results in different signaling consequences
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
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256
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and therefore different physiological responses in plants during cold stress During
stress phospholipids serve as a precursor for the generation of molecules which acts as
secondary messengers (Munnik et al 1998) Drought cold and salinity stresses
upregulates the expression of phospholipase C (PLC) which is a major enzyme
hydrolysing the phospholipids (Hirayama et al 1995 Kopka et al 1998) This increase
in expression of PLC results in the increase of cleavage of phosphotidylinositol
45-bisphosphate to produce diacylglycerol and inositol 145-triphosphate that acts as
key secondary messenger activating the protein kinase C by triggering Ca2+
release
The phospholipase D (PLD) has also proved to be involved in transducing the stress
signals by hydrolysing the phospholipids to phosphotidic acid The phosphotidic acid thus
formed mediates the ABA-induced stomatal closure in guard cells (Jacob et al 1999)
The role of calcium dependent protein kinases (CDPKs) in coupling inorganic signals to
specific phosphorylation cascade under stress has been reported (Urao et al 1994
Pei et al 1996 Hwang et al 2000) Overexpression of CDPK7 resulted in increased
tolerance to cold and osmotic stress in rice (Saijo et al 2000) In addition to the above-
mentioned sensors plants also use mitogen activated protein kinases (MAPKs) for
transducing the signals during abiotic stress Several MAPK modules (ie MAPKKK-
MAPKK-MAPK) that may be involved in abiotic stress signaling which are identified in
alfalfa rice (Kiegerl et al 2000) and tobacco (Yang et al 2001 Zhang amp Klessig 2001)
208
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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signaling in plants A mitogen-activated protein kinase pathway is activated by cold
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
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Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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52 MATERIALS AND METHODS
521 Screening of Putative Transformants (T0)-Tobacco and Tomato
5211 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the
putative transformants of tobacco tomato and WT plants as per Doyle amp Doyle (1990) as
mentioned in Chapter 4 (4212) The DNA was used as the template for PCR using AFP
internal primers The PCR products were resolved in 14 agarose gel and stained with
ethidium bromide The bands were visualized and documented The positive plants were
used for Southern analysis
5212 Isolation of Genomic DNA from Transgenic Plants for Southern Blot
Analysis- The genomic DNA from wild type tomato tobacco and transgenic lines
(tomato and tobacco) were isolated according to Michiels et al (2003) Around 1g of
young leaves were powdered well using liquid Nitrogen in a chilled mortar and pestle
The powdered samples were transferred to a 50mL sterile oak ridge tube containing
15mL of preheated extraction buffer (100mM Tris pH 8 14M NaCl 20mM EDTA pH
80 finally 2PVP and 02 β-mercaptoethanol were added freshly just before the use)
The samples were incubated at 60degC for 1h with occasional mixing to avoid aggregation
of the homogenate The samples were then centrifuged at 14000g for 10 min at 20degC
The supernatant was carefully transferred to a new tube and equal volume of Chloroform
Isoamyl alcohol (241) was added and vortexed thoroughly The samples were again
centrifuged at 14000g for 10 min at 20degC The extraction step was repeated with
chloroform isoamylalcohol (241) twice to get clear aqueous phase The aqueous phase
was mixed with 23 volume of ice-cold isopropanol by inversion and incubated at 25degC
overnight to precipitate the nucleic acids The samples were centrifuged at 14000g for
209
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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Use the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
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Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
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system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
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83-116
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144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
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plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
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Biologia Plantarum 546-12
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1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
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18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
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13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
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of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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10 min at 20degC and the supernatant were carefully removed To the pellet 15mL wash
solution (10mM ammonium acetate and 70 ethanol) was added and centrifuged at
14000g for 10 min at 20degC The washing step was repeated twice The supernatant was
removed and the pellet was air-dried resuspended in 1mL sterile TE buffer RNase A
(Fermentas Inc Maryland USA) was added to a final concentration of 10microgmL and the
samples were incubated at 37degC for 30 min After the incubation period 1mL of 25241
Phenol Chloroform Isoamylalcohol was added and the tubes were inverted several
times to get an emulsion The tubes were centrifuged at 14000g and the supernatant was
transferred to a fresh tube and twice the volume of absolute alcohol and 110 volume of
25 M sodium acetate were added and mixed well The tubes were incubated overnight at
-20degC for precipitation The DNA was pelletted at 14000g for 10 min at 4degC The pellet
was washed twice with 70 ethanol and the pellet obtained was air-dried The DNA was
resuspended in 100microL of nuclease free water The integrity of DNA was checked in 1
agarose and quantified in a Shimadzu 1601 UV‐Visible spectrophotometer (Shimadzu
Tokyo Japan)
52121 Restriction Enzyme Digestion of Genomic DNA and Agarose Gel
Electrophoresis for Southern Blot- The genomic DNA was digested with Xba I and Sac
I (Fermentas Inc Maryland USA) for Southern hybridisation The genomic DNA used
for the transgenic lines used for the Southern blot are shown in the table 51
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
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or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
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Use the
Text Box
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change the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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Plant Cell 122247-2258
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
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Plant Physiology 971265-1267
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Plant Physiology 901009-1014
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The EMBO Journal 156564-6574
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Expression profile of oxidative and antioxidative stress enzymes based on ESTs
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suspension cells Plant Physiology 130999-1007
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-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
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acclimation Journal of Plant Physiology 155762-768
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transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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signaling pathways Current Opinion in Plant Biology3217-223
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72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
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freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
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Genetic Systems 81349-354
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expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
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Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
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of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
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Springer Dordercht The Netherlands
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
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An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
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characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Sl No Sample Line Number
01 WT tobacco WT
02 Tobacco AFP 32
03 Tobacco AFP 41
04 WT tomato WT
05 Tomato AFP 1
06 Tomato AFP 3
Table 51- WT and Transgenic Lines of Tobacco and Tomato used for Southern Blot
The restriction enzyme digestion reaction mix was made as mentioned the table 52
Sl No Components Volume
01 Genomic DNA 20 microg (10 microL)
02 Digestion buffer 10X 5microl
03 Sterile water 21microL
04 Enzyme (10UμL) 2 + 2 microL
Total 50 microL
Table 52- Restriction Enzyme Digestion Reaction Mix used for Southern Blot
The samples were briefly centrifuged and incubated at 37degC in a waterbath
overnight for complete digestion The digestion product (5microL) was loaded and analysed
for the digestion in the agarose gel The digested genomic DNA from all transgenic lines
along with negative control (digested genomic DNA from WT plants) and positive
control (PCR product AFP) was loaded in 15 agarose gel Running buffer (1XTBE)
containing ethidium bromide was used The samples were resolved at 20V for overnight
The gel was photographed with a UV scale for determining the size
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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52122 Agarose Gel Treatments prior to Southern Hybridisation
521221 Depurination of Agarose Gel- The agarose gel was washed in depurination
solution (02N HCl) for 20 min The gel tray was placed in a gel rocker with slow speed
The gel was washed twice with deionised water after depurination
521222 Denaturation of Agarose Gel- The gel was incubated in denaturation solution (15 M
NaCl + 05M NaOH) for 15 min twice The gel was washed with deionised water
521223 Neutralisation of Agarose Gel- The gel was washed in neutralisation
solution (15M NaCl + 05M Tris HCl pH 72 1mM EDTA) for 15 min The gel was
washed with deionised water and was once again treated with neutralisation solution for
another 15 min The gel was finally washed with deionised water
521224 Transfer of DNA from Gel to Membrane- The DNA was transferred to the
positively charged nitrocellulose membrane (Biotrace NT Pall Life Sciences USA) by
capillary transfer in 20X SSC (3M NaCl + 03M Na3C6H5O7 + 1mM EDTA pH 70)
blotting buffer The transfer was left for overnight After the transfer the membrane was
briefly washed in 2X SSC in order to remove residual agarose adhering to the membrane
and the membrane was dried at room temperature The membrane was crosslinked by
exposing to UV light (120mJ) for 3 min
521225 Pre-hybridisation Probe Preparation and Hybridisation- The membrane
was incubated for 2h in prehybridisation solution (6X SSC 5X Denhardtlsquos solution
[100X Denhardtlsquos solution containing 2 wv BSA 2 wv Ficoll Type 400 2 wv PVP]
50 formamide 05 SDS) with 150μL of 10mgml denatured salmon sperm DNA The
membrane was incubated at 42degC in a rotating hybridisation chamber
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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the recrystallization of ice Cryobiology 3223-34
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Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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521226 Probe Preparation- The biotin labelling and detection kit (Fermentas Inc
Maryland USA) was used for labelling the probe and detected using chemiluminsecent
method The probe was labelled with Biotin as per the manufacturerlsquos instruction Briefly
1μL of biotin labelling mix was added along with the PCR reaction using AFP specific
primers The same condition was followed for PCR as mentioned in Chapter 4 (4213)
The probe was denatured at 95degC for 10 min and immediately placed in ice
521227 Hybridisation- After the prehybdrisation fresh hybdrisation solution was
added (same as prehybridisation solution except Salmon sperm DNA) and 10μL of
denatured probe was added to the hybridisation solution The hybridisation was carried
out for 12h at 42degC in a rotating hybridisation chamber
521228 Detection- After the hybridisation the membrane was washed twice in 2X
SSC + 01 SDS at 42degC for 10 min followed by two washes with 01 SSC + 01
SDS at 65degC for 10 min The membrane was partially dried by placing between the 2
Whatmann filter papers The detection was carried out according to the manufacturerlsquos
instruction The membrane was washed with 30mL of 1X Washing buffer for 5 min at
room temperature with moderate shaking The membrane was then incubated in blocking
solution for 30 min at room temperature with moderate shaking The membrane was then
washed twice with detection buffer for 10 min each After the second wash 20mL of
detection buffer was added and freshly prepared substrate solution was added over the
membrane The membrane was incubated at room temperature until a very specific blue
to pink insoluble precipitate forms on the membrane (colour development) representing
the probe specific bands
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Network India
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13321-25
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486-492
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Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
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United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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522 Analysis of T1 Transgenic Plants
5221 Segregation Analysis of Transgenic AFP Plants (T1)- The seeds AFP and WT
tobacco plants were inoculated in half strength MS media supplemented with 75mgL
Kanamycin Eighteen seeds were inoculated in each plates and the germination rate was
determined to check the segregation
5222 Germination (T1 Transgenic lines) - The seeds of T0 plants of tobacco and tomato
were sowed (in commercially available soil mix in pots) The germinated plants were
maintained in controlled conditions at 24degC with 168h lightdark The plants were nourished
with 110 strength sterile MS media daily The T1 plants were used for subsequent studies
52221 Genomic DNA Isolation and PCR- Genomic DNA was isolated from the T1
transgenic AFP and WT tobacco as mentioned in the Chapter 4 (4212) The DNA was
used as the template for PCR using AFP and nptII specific primers The nptII specific
primers are as follows
nptII Fw- 5lsquo- GTG GAG AGG CTA TTC GGC TA - 3lsquo
nptII Rev- 5lsquo- CAC CAT GAT ATT CGG CAA G-3lsquo
The PCR programme for nptII are as follows an initial denaturation at 94degC for
5 min followed by denaturation at 94degC for 1 min annealing at 58degC for 1min extension
at 72degC for 1 min for 30 cycles and a final extension at 72degC for 7 min
5223 Cold Stress Treatment- The different transgenic lines of tobacco (AFP5 AFP13
AFP 32 AFP 37 and AFP41) and WT tobacco plants were given cold stress at 4degC for
48h Young leaf samples were collected every 24h and all the samples were frozen in
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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point You
can
position the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
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Biologia Plantarum 546-12
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1213-15
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Arabidopsis Plant Physiology 127918-927
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Physiologia Plantarum 9311-18
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79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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liquid Nitrogen immediately until the isolation of RNA After 48h of stress the plants
were reverted to the normal condition and samples were collected after 24h (recovery
period)
The expression of AFP in tomato transgenic AFP plants was studied Three
transgenic lines (AFP1 AFP3 and AFP3) along with WT plants were given cold stress
for 48h Samples were harvested at 0h and 48h and stored in liquid Nitrogen until further
process
In order to determine the expression of AFP in different tissues of transgenic tobacco
plants one of the tobacco line AFP 41 and WT tobacco were given cold stress for 48h
Different tissues like root stem mature leaves young leaves and flowers were harvested at
every 24h and frozen in liquid nitrogen immediately until isolation of RNA The expression
of AFP in transgenic and wild type plants were determined by semi-quantitative Reverse
Transcriptase PCR (sq RT-PCR) All the sq RT-PCR was repeated twice for confirmation
The plant phenotype under stress was monitored carefully Comparative analyses
of the various molecular biochemical physiological and phenotypic parameters related
to cold stress were assayed in both transgenic and non-transformed plants under normal
(non-stress) and stressed conditions
5224 Expression Analysis of Antioxidant Enzymes and Signaling Molecules- Two
weeks old transgenic lines of AFP 32 AFP 41 and WT tobacco plants were given cold
stress at 4degC for one week Samples were harvested at 0h after 3 days and 7 days of
stress The samples were immediately frozen in liquid Nitrogen and stored at -80degC until
further process
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
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Use the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
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83-116
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plants Plant Journal 4215-223
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De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
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1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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52241 sq RT-PCR- RNA was isolated from all the samples using Qiagen RNeasy
RNA isolation kit (Qiagen Hilden Germany) as per manufacturerlsquos instruction
(The same protocol was followed as mentioned in Chapter 2 - 2221) On column DNase
treatment was performed using RNase-Free DNase (Qiagen Hilden Germany) according
to manufacturerlsquos instruction
52242 Quantification and Electrophoresis of RNA- The yield of the isolated RNA
was determined in Nanodrop 2000c (Thermo scientific Wilmington USA) The 260280
ratio was also determined The integrity of RNA was checked in denaturing agarose gel
The same protocol was followed as mentioned in Chapter 3 - 3231
52243 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using
RETROscriptreg Kit - Reverse Transcription for RT-PCR (Ambion Inc USA) as
mentioned in Chapter III- 3232
52244 Expression Analysis of AFP Antioxidative Enzymes and Signaling
Molecules in Transgenic Tobacco by sq RT-PCR- The first strand cDNA was diluted
110 times in nuclease free water The PCR was performed using ―Ready to Go PCR
beads (GE healthcare NJ USA) using AFP specific primers The PCR programme was
same as mentioned in the Chapter 3-324 Ntabacum β- tubulin was used as the
reference for the PCR reaction to normalize the gene expression The samples were
resolved using 14 agarose gel and the gel was stained for 30 min with ethidium
bromide (80ngmicroL) and destained in water for 10 min The bands were visualized and
documented The cDNA from the two transgenic lines (AFP 32 and AFP 41) and WT
from different treatment times (0h 3 days and 7 days stress) were used for the expression
analysis of antioxidant enzymes and signaling molecules The cDNA were diluted as
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
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Physiologia Plantarum 9311-18
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18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
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stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
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53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
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vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
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62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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mentioned above and PCR was performed using PCR beads The primer sequence and
PCR conditions for the antioxidant enzymes and signaling molecules are shown in the
table 53
523 Estimation of Proline in Transgenic Plants- Transgenic tomato tobacco and WT
plants were given cold stress at 4ordmC for 48h Plant samples (100mg) were extracted using 3
sulphosalicylic acid and filtered through Whatman No1 filter paper To 2mL of filtrate 2mL of
glacial acetic acid and 2mL of Ninhydrin reagent were added and incubated in a boiling water
bath for 1h The reaction was terminated by incubating the mixture in an ice- bath Toluene
(4mL) was added and vortexed vigorously for 20 s and incubated at 23ordmC for 24h The toluene
layer was separated and absorbance was measured at 520 nm (Bates et al 1973) The
experiment was repeated thrice with different samples under the same conditions
524 Estimation of Total Soluble Sugars in Transgenic Plants- Transgenic tomato
tobacco and WT plants were given cold stress at 4ordmC for 48h Plant samples (100mg)
were grounded well in liquid Nitrogen Ethanol (15mL of 75) was added and incubated
overnight in an orbital shaker at 150 rpm The samples were centrifuged at 20000g for
5 min Anthrone reagent (1mL of 02 ) was added to the supernatant and incubated for
1h at 100ordmC The absorbance was measured at 625nm (Li et al 2004) The experiment
was repeated thrice with different samples under the same conditions
525 Electrolytic Leakage Assay- Electrolytic leakage or Ionic leakage assay was
performed according to Xu et al (2005) The control and transgenic plants were given cold
stress at 4ordmC and the leaf discs of equal area were collected at 0h 24h and 48h The discs
were equilibrated in deionised water for 4h and initial conductivity was measured The
217
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
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or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
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Use the
Text Box
Tools tab to
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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samples were autoclaved to get all the solutes from cells and the final conductivity was
measured
The relative conductivity () was calculated using the formula
Relative Conductivity () =
The experiment was repeated thrice with different samples under the same conditions
Sl No Gene Primer sequence PCR condition 01 Catalase Fw 5lsquondash ACA AGA TGC TTC AAA CTC GT ndash3lsquo
Rev 5lsquo ndash AGT GAT TGT TGT GAT GAG CA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
58ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
02 SOD Fw 5lsquo- GGT CAC ATT AAC CAC TCG AT ndash 3lsquo
Rev 5lsquo ndash AGT TAG TGT CGA TAG CCC AAndash3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
03 APX Fw 5lsquo ndash TAC CTA TGA TGT GTG CTC CA - 3lsquo
Rev 5lsquo- CCT CCA GTA ACT TCA ACA GC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 sec
30 cycles 72ordmC 10 min
04 GPX Fw 5lsquo ndash TGG CCT GAC TAA TTC AAA CT-3lsquo
Rev 5lsquo- CTG GAT CTC TTC AAT GCT TC - 3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
05 GST Fw 5lsquo- AAT CAC CCA ATA CAT TGC TC - 3lsquo
Rev 5lsquo- CAT CTT CTT TGG ATC TTG GA ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
56ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
06 PLA Fw 5lsquo ndash AGC CA GTG GT TGA ACT AGA A - 3lsquo
Rev 5lsquo ndash TGA AAG GTA GAG CCA CTG TT- 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
07 PLC Fw 5lsquo- ATG GTG GTT GTG GAT ATG TT - 3lsquo
Rev 5lsquo- AAA TAC GGT CAC CTT CAA TG ndash 3lsquo
94ordmC 5 min 94ordmC 30 s
55ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
08 AOX Fw 5lsquo- GGT ACT GAA TGG AAA TGG AA ndash3lsquo
Rev 5lsquo ndash ATA AGC GAA TTT GTC CAA GA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
29 cycles 72ordmC 10 min
09 MAPKK Fw 5lsquo-ACC TTC GAA TTT GCT AAT CA -3lsquo
Rev 5lsquo- TAA GTG CCG ACA AAG GTA TT -3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
10 P5CS Fw 5lsquo-ATA TAT CGG CTG GCA TTA AA ndash 3lsquo
Rev 5lsquo- GGA GAT GAT GAT TTC TCC AA-3lsquo
94ordmC 5 min 94ordmC 30 s
53ordmC 30 s 72ordmC 30 s
30 cycles 72ordmC 10 min
11 β-Tubulin Fw 5lsquo- CTG CGG AAA CTT GCT GTG AA-3lsquo
Rev 5lsquo- AAG TGG AGC AAA CCC AAC CA-3lsquo
94ordmC 5 min 94ordmC 30 s
60ordmC 30 s 72ordmC 30 s
28 cycles 72ordmC 10 min
Table 53ndash Primer Sequences and PCR Conditions for Antioxidant Enzymes
Signaling Molecules
X 100
Initial conductivity
Final conductivity
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
the
document
or the
summary of
an
interesting
point You
can
position the
text box
anywhere
in the
document
Use the
Text Box
Tools tab to
change the
formatting
W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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526 Phenotype Analysis- The transgenic lines of tobacco (AFP32 and AFP 41) and WT
plants were germinated in sterile soil under controlled conditions mentioned above
Ten days old seedling was transferred to cooling incubator at 4degC The growths of the
plants were measured for three weeks The ability of WT and transgenic lines to recovery
(after stress) was also studied The experiment was repeated twice under the same
conditions
219
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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interesting
point You
can
position the
text box
anywhere
in the
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Use the
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Tools tab to
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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of Experimental Botany 582969-2981
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88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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53 RESULTS AND DISCUSSION
531 PCR and Southern Analysis of Transgenic Tobacco and Tomato plants- In an
attempt to enhance the cold tolerance Carrot AFP was genetically transformed to tobacco
and tomato The T0 putative lines of tobacco and tomato were screened using AFP primers
Out of 19 lines of tobacco screened 15 lines were positive for PCR (fig 52 53) In case of
tomato only 5 lines were screened 3 plants were positive in PCR for AFP Southern blot
analysis was performed using the AFP PCR product as a probe for two PCR positive
plants of tobacco and tomato As the aim of the Southern blot was only to study the AFP
integration (not copy number) the genomic DNA was digested with Xba I and SacI
which flank the AFP gene in the T-DNA [fig 54 (a)] The hybridisation signal
corresponding to the full length of the gene (11kb) was observed [fig 54(b) 54(c)]
This shows that the AFP was integrated to the plant genome in all the 4 plants analysed
Figure 52ndash Isolated Genomic DNA from Transgenic Tobacco Tobacco and WT Plants
Lane M- Marker DNA Lane 1- 8- DNA from putative tobacco plants
Lane 9- 17- DNA from putative tomato plants Lane 18 19- WT tobacco and tomato plants
WT Tomato DNA Tobacco DNA
250bp
500bp
10kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Genomic DNA
220
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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summary of
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interesting
point You
can
position the
text box
anywhere
in the
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Use the
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Tools tab to
change the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Physiologia Plantarum 9311-18
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13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
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Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
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22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
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plantagineum Plant Cell 12111-123
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24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
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Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
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United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
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temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
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genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
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of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 53ndash PCR Amplification of Transgenic Tobacco and Tomato Plants
Lane M 24- Marker DNA Lane 1- 10- AFP amplicons from tobacco plants
Lane 11- 20- AFP amplicons from tomato plants Lane 21- WT tobacco
Lane 22- WT tomato Lane 23- AFP from binary vector (positive control)
10kb
3kb
200bp
100bp
250bp
500bp
M 1 2 3 4 5 6 7 8 9 10 111213 14 1516 1718 19 2021 22 23 24
221
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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Use the
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
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32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
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2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
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the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 54- Southern Blot Analysis of Transgenic Plants with AFP
Fig 54 (a) ndash T- DNA region of pBI121-AFP- 11kb AFP PCR product was used as probe
Fig 54 (b) ndash Digestion of tobacco and tomato DNA with XbaI and SacI for Southern blotting
Lane M- Marker DNA Lane 1- AFP PCR product (positive control) Lane 2- WT tobacco
DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5- WT tomato DNA Lane 6 amp 7-
Tomato AFP 1 amp AFP 3
Fig 54 (c) - The Southern analysis of transgenic tobacco and tomato plants Lane 1- AFP PCR
product Lane 2- WT tobacco DNA Lane 3 amp 4- Tobacco AFP 32 Tobacco AFP 41 Lane 5-
WT tomato DNA Lane 6 amp 7- Tomato AFP 1 amp AFP 3
AFP nptII CaMV 35S LB RB
SacI XbaI
11kb
54 (a)
54 (b)
Tomato Tomato Tobacco Tobacco
54 (c)
11kb
100b
p
1kb
10kb
M 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
quote from
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interesting
point You
can
position the
text box
anywhere
in the
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Use the
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Tools tab to
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
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88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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532 Segregation Analysis of Transgenic Tobacco Plants- From the analysis we found
that transgenic plants showed Mendelian pattern of inheritance Out of the 18 seeds
inoculated 14 seeds germinated in half strength MS media with Kanamycin (fig 55) Few
WT seeds germinated initially in the selection media however they failed to grow later
Figure 55 - Transgenic Tobacco Line AFP 32 Inoculated in Half Strength MS Media
with Kanamycin to Study the Segregation Analysis
533 Confirmation of T1 Transgenic Tobacco and Tomato by PCR- PCR was
performed for T1 transgenic tomato and tobacco plants with AFP primers The expected
size bands (11kb) were observed in both tobacco and tomato transgenic plants However
the number of PCR positive lines was less for tomato The tobacco plants were also
confirmed with nptII primers that gave bands at expected size (fig 56 57 58 59 510)
Figure 56ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 8 - DNA from T1 transgenic tobacco lines
Tobacco
DNA e a
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W
T
10kb
100bp
Genomic DNA
M 1 2 3 4 5 6
7
200bp
223
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Plant Physiology 130618-626
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induced RD29A and RD29B promoters by constitutively active Arabidopsis
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31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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32336-348
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38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
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42 Knight H (2000) Calcium signaling during abiotic stress in plants International
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
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stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
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53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
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generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
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level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
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Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
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60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
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peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
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reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
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induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
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the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
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of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
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water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
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National Academy of Sciences of United States of America 98741-746
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acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 57ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- WT DNA Lane 3- 9- AFP
amplicons from T1 tobacco lines Lane 10- PCR amplicon from Carrot DNA
Figure 58ndash PCR Amplification of Transgenic Tobacco T1 Lines with nptII Primers
Lane M- Marker DNA Lane 1- Positive control Lane 2- 6-nptII amplicons from T1
tobacco lines Lane 7- WT DNA
600bp
100bp
750bp
1kb
M 1 2 3 4 5 6 7
11kb
M 1 2 3 4 5 6 7 8 9 10
10kb
200bp
100bp
1kb
224
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 59ndash Isolated Genomic DNA from WT and Transgenic Tobacco T1 Lines
Lane M- Marker DNA Lane 1- WT DNA Lane 2- 6- DNA from T1 tomato lines
Figure 510ndash PCR Amplification of Transgenic Tobacco T1 Lines with AFP Primers
Lane M- Marker DNA Lane 1-WT DNA Lane 2- 5- AFP amplicons from T1 tomato
lines Lane 6- PCR amplicon from Carrot DNA
10 kb
1kb
100bp
11kb
M 1 2 3 4 5 6
WT Tomato DNA
10kb
M 1 2 3 4 5 6
1kb
100bp
Genomic DNA
225
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
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Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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32336-348
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38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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42 Knight H (2000) Calcium signaling during abiotic stress in plants International
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potato Plant Physiology 116239-250
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
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stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
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53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
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generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
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Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
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Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
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vesicles Biochimica et Biophysica Acta 1021133-140
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Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
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60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
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peroxide Plant Cell 665-74
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induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
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protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
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Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
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General Genetics 224331-340
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Physiology 100115-119
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the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
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of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
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protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
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National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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534 AFP Expression Analysis by sq RT-PCR
5341 RNA Quantification and cDNA Synthesis ndash The RNA was quantified from all
the samples and the integrity of the RNA was checked in denaturing gel Equal amount of
RNA (2microg) from all the samples were used for the synthesis of cDNA The yield of RNA
and the purity is given in the following tables (54 55 56)
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 3004 212
02 AFP 5 (Control) 5541 216
03 AFP 13 (Control) 3815 214
04 AFP 32 (Control) 5173 213
05 AFP 37 (Control) 8052 21
06 AFP 41(Control) 8165 211
07 WT (24 hour stress) 4581 216
08 AFP 5 (24 hour stress) 5479 211
09 AFP 13 (24 hour stress) 9334 214
10 AFP 32 (24 hour stress) 6330 209
11 AFP 37 (24 hour stress) 7102 204
12 AFP 41(24 hour stress) 6921 21
13 WT (48 hour stress) 4872 212
14 AFP 5 (48 hour stress) 5227 216
15 AFP 13 (48 hour stress) 7432 209
16 AFP 32 (48 hour stress) 5643 212
17 AFP 37 (48 hour stress) 2985 208
18 AFP 41(48 hour stress) 8639 212
19 WT root (24 hour recovery) 4877 212
20 AFP 5 (24 hour recovery) 5192 215
21 AFP 13 (24 hour recovery) 3984 21
22 AFP 32 (24 hour recovery) 7032 212
23 AFP 37 (24 hour recovery) 7652 214
24 AFP 41 (24 hour recovery) 6283 211
Table 54ndash Quantification of RNA from WT and Transgenic Tobacco Lines during
Cold Stress
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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the recrystallization of ice Cryobiology 3223-34
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Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
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44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
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alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Expression of the transgene was assessed using sq RT-PCR in (5)- T1 lines of tobacco
and (3)- T1 lines of tomato that had been exposed to low temperature stress
Sq RT-PCR confirmed the expression of the transgene (AFP) in all the transgenic lines
studied in T1 generation However the expression of gene was same throughout the stress
period (fig 510 511) There was also no change in the expression levels of AFP during the
recovery period constitutive CaMV 35S promoter drives the gene The Thermal Hysteresis
Activity (THA) of the AFP expressed in transgenic tobacco plants plays a key role in
conferring low temperature tolerance Ice recrystallization occurs spontaneously when plants
are exposed to low temperature for prolonged periods (Knight et al 1995) The carrot AFP
with THA in the transgenic tobacco plants may inhibit ice growth and recrystalization hence
it prevents the plant cells from the cold damage Among the different transgenic lines tested
Tobacco AFP 32 and Tobacco AFP 41 showed higher transcript levels in sq RT-PCR
analysis which may be due to the different integration sites in the tobacco genome Hence
these two transgenic lines were chosen for detailed analysis in following works
Figure 511- sq RT- PCR in T1 Tomato Lines
Three different tomato lines and WT plants were given cold stress at 4degC for different time
intervals β-tubulin was used as the reference gene for sq RT-PCR
C 48h 48 hour stress (48h) Control (C)
AFP
β- Tubulin
RNA
AFP1 AFP3 AFP3 AFP1 AFP3 AFP3 WT WT
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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potato Plant Physiology 116239-250
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Biochemistry 31585-89
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Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
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generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
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Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
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Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
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vesicles Biochimica et Biophysica Acta 1021133-140
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The EMBO Journal 156564-6574
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60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
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409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
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seedlings during stress Nature Biotechnology 15988-991
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phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
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single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
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transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
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72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
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freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
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reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
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glutathione-S-transferase Proceedings of the National Academy of Sciences of
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Physiology 100115-119
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expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
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Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
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of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
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National Academy of Sciences of United States of America 98741-746
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Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
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An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 512- sq RT- PCR in T1 Tobacco Lines
Five different transgenic tobacco lines and WT plants were given cold stress at 4degC at different
time intervals β-tubulin was used as the reference gene
It was also found in tobacco that the AFP gene is ubiquitously expressed in
different vegetative tissues and reproductive organ like flower of the transgenic plants
The expression levels of AFP appeared to be almost same in all the tissues examined
Smallwood et al (1999) reported that the transcript accumulation of AFP in carrot was
high in cold acclimated roots followed by leaf and stem A novel chitinase with antifreeze
24 hour recovery
48 hour stress
24 hour stress
Control
β- Tubulin
β- Tubulin
β- Tubulin
β- Tubulin
RNA
AFP
AFP
AFP
RNA
RNA
RNA
WT AFP5 AFP14 AFP32 AFP37 AFP41
AFP
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
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88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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activity was recently characterised from Chimonanthus praecox and its expression levels
were highest in corolla followed by leaf root and bark tissue (Zhang et al 2011)
This ubiquitous expression level of AFP in transgenic plants can be attributed to the
activity of constitutive CaMV 35S promoter With the increase in isolation and
characterisation of inducible and tissue specific promoter it may be possible in future to
tune the expression of AFP to obtain the desired level of cold tolerance avoiding any
undesirable consequences of constitutive expression In addition to this further studies in
the field conditions after getting all the biosafety approval
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT root (Control) 1230 216
02 WT mature leaves (Control) 6656 216
03 WT young leaves (Control) 12004 215
04 WT stem (Control) 4801 21
05 WT flower (Control) 6866 215
06 AFP 41 root (Control) 1484 208
07 AFP 41 mature leaves (Control) 4562 217
08 AFP 41 young leaves (Control) 9087 204
09 AFP 41 stem (Control) 6641 219
10 AFP 41 flower (Control) 4523 213
11 WT root (24 hour stress) 2720 211
12 WT mature leaves (24 hour stress) 5321 214
13 WT young leaves (24 hour stress) 13721 215
14 WT stem (24 hour stress) 5546 199
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Network India
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Plant Physiology 1071049-1054
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system of eastern white pine needles Plant Physiology l98501-508
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12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
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United States of America 923903-3907
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29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
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Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
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understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
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oxygen gene network of plants Trends in Plant Science 9490-498
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components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
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generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
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level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
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Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
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Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
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peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
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phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
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transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
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McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
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Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
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induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
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alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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Physiology 145148-152
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insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
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expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
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protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
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water stress- Evolution of osmolyte systems Science 2171214-1217
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kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
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acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
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characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
15 WT flower (24 hour stress) 6230 215
16 AFP 41 root (24 hour stress) 2840 209
17 AFP 41 mature leaves (24 hour stress) 4232 212
18 AFP 41 young leaves (24 hour stress) 9286 211
19 AFP 41 stem (24 hour stress) 5992 218
20 AFP 41 flower (24 hour stress) 5440 214
21 WT root (48 hour stress) 1423 208
22 WT mature leaves (48 hour stress) 6234 207
23 WT young leaves (48 hour stress) 11742 215
24 WT stem (48 hour stress) 6285 212
25 WT flower (48 hour stress) 5670 202
26 AFP 41 root (48 hour stress) 1192 208
27 AFP 41 mature leaves (48 hour stress) 3998 211
28 AFP 41 young leaves (48 hour stress) 14571 206
29 AFP 41 stem (48 hour stress) 7335 218
30 AFP 41 flower (48 hour stress) 3893 217
Table 55- Quantification of RNA from Different Organs in WT and Transgenic
Plants After Cold Stress
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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Cell 11691-706
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transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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freezing tolerance Plant Cell amp Environment 26523-535
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Genetic Systems 81349-354
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expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
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255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
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Springer Dordercht The Netherlands
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567-575
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An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
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in Science 6520-527
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characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 513- sq RT- PCR in Different Tissues of T1 Tobacco Lines
Transgenic tobacco line 32 and WT plants were given cold stress at 4degC RNA was isolated from
different organs like Root (R) Stem (S) Mature leaves (ML) Young leaves (YL) and Flower (F)
cDNA was synthesised from 2μg of RNA from all the samples and used as template for PCR to
determine the transcript accumulation of AFP in different organs β-tubulin was used as the
reference gene
Freezing occurs first in the extracellular space in a plant where the fluids in the
cell have a higher freezing point than the fluids present inside the cells (Atici amp
Nalbantoglu 2003) This results in water loss inside the cell due to osmosis thus causing
β- tubulin
β- tubulin
β- tubulin
AFP
RNA
AFP
RNA
AFP
RNA
48 hour stress
24 hour stress
Control
R S ML YL F R S ML YL F
WT AFP 32
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Proline induces the expression of salt stress-responsive proteins and may improve
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Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
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88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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cell dehydration All known AFPs from the plant have shown limited effect on thermal
hysteresis therefore it is assumed AFPs are primarily involved in ice recrystallization
inhibition (IRI) In vitro characterization of AFPs has demonstrated their IRI activities
(Griffith et al 1992 Sidebottom et al 2000 John et al 2009) suggesting their critical
role in conferring freezing tolerance in plants
The T1 transgenic lines of tobacco and tomato were germinated and studied for
response to cold stress by a number of important abiotic stress related molecular
biochemical and physiological parameters which may be regulated due to the integration
of the AFP gene into the genome of tobacco and tomato lines
The expression levels of critical genes implicated in oxidative metabolism and
signaling enzymes were studied by sq RT-PCR in order to gain insights into the
regulation of the antioxidant and signaling pathways in transgenic plants with AFP
The transcript levels of endogenous genes in tobacco were monitored before and after
cold stress treatment that includes 5 antioxidant enzymes (NtAPX NtGPX NtCAT
NtGST NtSOD) and 4 signaling enzymes (NtMAPKK NtPLA NtPLC and NtAOX) and
delta 1-pyrroline-5-carboxylate synthetase (NtP5CS) which is involved in the synthesis
of proline (fig 514 515) These genes or homologues have been often shown to be
involved in cold stress response No significant variation in transcript levels were
observed in both the transgenic lines (at all time points) however the transcript level of
all the antioxidant enzymes were high (except catalase) at all time points in the WT
plants The expression of signaling candidates MAPKK AOX were also high in WT
plants The transcript abundance of PLA and PLC were high in one of the transgenic line
(AFP 32) studied during stress This change in expression level between different
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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accumulation of active oxygen species during the hypersensitive reaction of bean
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2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
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Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
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(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
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79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
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genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
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alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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transgenic lines can be attributed to the difference in the site of integration of the AFP in
the plant genome The expression of P5CS was also high in the WT plants Higher
expression levels of these genes in WT plants may probably provide more chaperones for
various substrates the up-regulation of antioxidant enzymes for scavenging free radicals
increase in synthesis of osmolytes signaling for activation of transcription factors for
induction of cold responsive genes etc which are known mechanisms in plants to
sustain growth during low temperature stress though in our studies it is not the case
Sl No Sample at different time point of stress RNA concentration (ngmicroL) 260280 ratio
01 WT (Control) 10393 211
02 WT (3 days stress) 3097 213
03 WT young leaves (7 days stress) 5507 212
04 AFP 32 (Control) 4602 214
05 AFP 32 (3 days stress) 9007 211
06 AFP 32 (7 days stress) 6084 209
07 AFP 41 (Control) 9009 212
08 AFP 41 (3 days stress) 3911 214
09 AFP 41 stem (7 days stress) 7568 212
Table 56- Quantification of RNA from WT and Different Transgenic Tobacco Lines
during Cold Stress
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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Plant Physiology 1071049-1054
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20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
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486-492
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Characterization of alternative oxidase (AOX) gene expression in response to
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Environment 1211-215
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28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
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29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
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Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
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33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
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2164-11-73
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of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
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Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
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America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
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40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
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60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
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alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
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Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
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glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
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the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
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kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
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responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
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An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Figure 514- sq RT-PCR for Antioxidant Enzymes in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC at different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and used as template for
PCR to determine the transcript accumulation of various antioxidant enzymes β-tubulin was used
as the reference gene
Exposure to low temperature may result in the increased generation of free radicals in
higher plants (Okuda et al 1991 Prasad et al 1994) These ROS not only attack the plants
cellular components but also deliver signals for perceiving the changed environment
(Fridovich 1991) The relationship between antioxidant capacity and low temperature
tolerance in plant was reported by Foyer et al (2001) Low temperature exposure of winter
cereals showed an increased tolerance to oxidative stress (Bridger et al 1994) The activities
of several antioxidant enzymes are also increased during cold acclimation in wheat (Scebba
et al 1999) These data suggests that increase in tolerance to such stress is always
accompanied by increased expression of specific genes encoding different antioxidant
enzymes The expression of SOD were high in WT plants in our studies where as the
AFP 32 AFP 41 WT
C 3days 7days C 3days 7days C 3days 7days
SOD
APX
GPX
GST
Catalase
β- Tubulin
RNA
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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Physiology 1131177-1183
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255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
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both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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expression level was stable in both the transgenic lines tested during all time points
The MnSOD genes in plants are nuclear encoded and the protein is targeted to mitochondria
The up-regulation of gene in WT plants observed in this study suggests a protective
mechanism targeted to the mitochondria As the electron transport chain in mitochondria has
been shown to be very sensitive to the change in environmental temperatures the electrons
that are not transported to the final electron acceptor ie oxygen to produce water might be
used for the formation of superoxide ion (O2minus
) The operation of the mitochondrial
alternative pathway involving the expression of AOX at low temperature may also produce
more superoxide radicals The other forms of SOD ie CuSOD ZnSOD and FeSOD genes
are targeted to chloroplast suggesting that the constant expression of these genes may protect
chloroplasts from damage induced by superoxide during cold acclimation Apart from this
the APX also play a key role in chloroplasts in capturing the free radicals APX quickly
scavenges the peroxides at the thylakoid membrane and GSH-dependent DHAR that is
located in the stroma of chloroplasts which functions in the waterndashwater cycle for removing
peroxides from the cell The transcript level of APX were high in WT type plants during
stress and were indistinguishable in transgenic lines suggesting that chloroplast activity has
not affected in similar manner the transgenic lines on the exposure to cold temperature
The enzymes GPX and GR catalyze the oxidation of NAD(P)H to NAD(P)+ The expression
of GPX were little higher in WT plants after one week of stress once again in transgenic
lines we could not find any change These observations suggests GPX (along with GR
which we have not studied) might be involved in the production of NAD(P)+ which may be
incorporated into light energy capture (Vanlerberghe et al 1992) the oxidative portion of the
pentose phosphate pathway or glycolytic pathway These processes contribute to the net
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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-dependent protein kinase confers both cold and saltdrought tolerance
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74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
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79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
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88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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carbon assimilation and relative growth rates especially in WT plants The assimilated carbon
might be used as the backbone for the synthesis of sugars and other osmolytes which can
protect the cells from osmolysis during low temperature stress GSTs are the enzymes
involved in cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione
(GSH) to different substrates like xenobiotic compounds Plant GSTs are expressed in
response to various stresses such as metal stress oxidative stress phytohormone treatment
inorganic phosphate starvation etc (Takahashi amp Nagata 1992 Ezaki et al 1995 Richards
et al 1998) In Arabidopsis GST1 and GST11 were proved to be induced by cold and
Aluminum stress (Roxas et al 1997 Ezaki et al 2001) Expression analysis for rice
seedlings under abiotic stress conditions revealed that 20 different GST (isoforms) were
differentially expressed significantly under at least one of the abiotic stress conditions
studied (Jain et al 2010) The expression of catalase remained constant during cold treatment
in the WT and transgenic lines studied Eventhough from the reports it is evident that catalase
enhances chilling tolerance in rice tomato and other plants (Matsumura et al 2002
Wang et al 2005) in our case we found a stable expression level even in the WT plants
The enzyme is located in peroxisomes or glyoxysomes which is actively involved in the
scavenging hydrogen peroxide As mentioned above the peroxides are also quenched by the
APX present in either cytosol or chloroplast using reduced ascorbate (Allen 1995
Willekens et al 1997) It can be postulated that the peroxides formed in WT plants are
quenched by the APX in the thylakoid resulting in lack of substrate (peroxide) for catalase
Janda et al (2003) reported the expression of catalase was down regulated after 7 weeks of
cold acclimation in wheat which might be due to the increased activity of the plastid bound
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
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Plant Cell 122247-2258
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
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catalase expressed in transgenic rice can improve tolerance against low temperature
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Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
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oxygen gene network of plants Trends in Plant Science 9490-498
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components in plants Annual Review of Plant Biology 58459-481
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illumination Plant and Cell Physiology 211295-1307
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The EMBO Journal 156564-6574
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60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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APX Khedr et al (2003) also reported a reduction in catalase activity during the salt stress
in Pancratium maritimum
The results presented here suggest that genes encoding antioxidant enzymes may
play a pivotal role in cold response in partial protection of the WT plants All the genes for
antioxidant enzymes except catalase were upregulated during cold stress and the antioxidant
enzymes may be used to protect plant cells from oxidative stress resulting from exposure to
cold temperature However the constant expression of all the enzymes in both the transgenic
lines throughout the stress period may suggest that the plants are not experiencing any stress
(due to the presence of transgene) and hence the production of ROS might be less
Figure 515- sq RT- PCR of Signaling Molecules in T1 Tobacco Lines
Transgenic tobacco line 32 41 and WT plants were given cold stress at 4degC for different time
intervals cDNA was synthesised from 2μg of RNA from all the samples and was used as
template for PCR to determine the transcript accumulation of signaling molecules and enzymes
involved in the biosynthesis of Proline β-tubulin was used as the reference gene
PLC
PLA
AOX
MAPKK
RNA
P5CS
β- Tubulin
C 3days 7days C 3days 7days C 3days 7days
AFP 32 AFP 41 WT
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
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Plant Cell 122247-2258
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potato Plant Physiology 116239-250
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
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understanding and application Trends in Biotechnology 8358-362
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Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
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oxygen gene network of plants Trends in Plant Science 9490-498
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illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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MAP related kinases catalyze reversible phosphorylations reactions and are
important for relaying signals to various targets during stress They function via kinase-
phosphatase cascades which involve in sequential phosphorylation of a kinase by its
upstream kinase (Xiong amp Ishitani 2006) The expression of MAPKK was found to be
more in WT plants during stress which implies that the low temperature signals are
relayed to nucleus through MAPKK However in our study the expression of MAPKK
was found to be constant in both the transgenic lines at all time points We presume that
the transgenic lines are not experiencing the cold stress because of the presence of AFP
under constituitive promoter 35S and hence MAPKK would not have been activated
A MKK2 pathway was identified in Arabidopsis and reported to be involved in cold and
osmotic stress signal transduction A MAPK with specific involvement in drought and
salt stress is through p44MMK4 kinase identified from alfalfa (Jonak et al 1996) Xing
et al (2007) showed that over-expression of AtMEK1 in Arabidopsis showed increased
tolerance to drought or salt stress which indicates that AtMEK1 acts as a positive
regulator of drought and salt stress Some MAPK and MAPKK proteins have also been
shown to activate the Rd29A stress pathway in Arabidopsis (Hua et al 2006)
Plant mitochondria possess a unique respiratory pathway in addition to the main
cytochrome pathway The alternative pathway is a non-phosphorylating electron transport
pathway and the electron flow through the pathway reduces the molecular oxygen to
water without the conservation of energy in the form of ATP A variety of biotic and abiotic
stress conditions induce alternative oxidase (AOX) and steady state transcript levels of AOX
genes increased under low temperature stress (Ito et al 1997 Takumi et al 2002
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
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40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
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Plant Cell 122247-2258
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Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
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understanding and application Trends in Biotechnology 8358-362
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Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
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oxygen gene network of plants Trends in Plant Science 9490-498
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components in plants Annual Review of Plant Biology 58459-481
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Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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Fung et al 2006 Sugie et al 2006) The expression of AOX was high in WT plants at
all time points in our studies whereas the there were no significant change in transcript
accumulation in transgenic tobacco lines During cold stress the respiration capacity of
the alternative pathway is increased significantly in WT plants and this enhancement
seems to be coupled with the AOX transcript accumulation High levels of the AOX
protein and the alternative pathway partly would have lead to freezing tolerance of the
WT plants in the present study As the transgenic plants possibly were not experiencing
the effect of cold stress the respiration might occur through normal cytochrome pathway
due to which the alternative pathway might not have been activated hence AOX
transcript levels remains same in transgenic plants
Three classes of phospholipases PLD PLC and PLA2 are identified and studied
widely for their roles in lipid derived signalling pathways PLC and PLA2 are the
well-researched signaling enzymes in plant and animal systems PLC produces the
second messengers diacyl glycerol (DAG) inositol phosphate (IP) and PLA2 catalyzes
the rate limiting step in eicosanoid synthesis and regulation Phosphatidic acid was
reported to accumulate in response to cold treatment (Ruelland et al 2002) and water
deficit (Frank et al 2000) in plants and it is formed by the phosphorylation of
diacylglycerol by phospholipase C PLA2 in cold stress responses is involved in
remodelling of membrane phospholipids The activity of the plasma membrane
H+-ATPase is modulated by PLA2 (Palmgren amp Sommarin 1989 Palmgren et al 1990)
and changes in the properties of membrane transport proteins like plasma membrane
ATPase have been observed upon freezing stress (Yoshida amp Uemura 1990)
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
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induced RD29A and RD29B promoters by constitutively active Arabidopsis
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
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Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
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signaling in plants A mitogen-activated protein kinase pathway is activated by cold
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America 9311274-11279
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mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
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the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
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Botanical Bulletin of Academia Sinica 399-15
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phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
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Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
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Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
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components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
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illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
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70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
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The transcript accumulation of PLA and PLC were high in WT and AFP 32 at all time
points whereas expression levels of both the genes in AFP 41 were found to be slightly
higher at 0h and 7 day stress (at 3 days stress it was less) We could provide two possible
explanations for the upregulation of PLA and PLC in the transgenic lines as it cannot be
ruled out that PLA and PLC are upregulated (due to the presence of transgene) and it
may be involved in transmitting the stress signals The change in expression level of the
genes in AFP 41 could also be due to the insertional effect of the transgene in the plant
genome Drought or salt stress induced the upregulation of PLC isoforms in Arabidopsis
(Hirayama et al 1995 Kopka et al 1998) This increase in PLC expression could
contribute to increased cleavage of phosphotidylinositol 45-bisphosphate (PIP2) to
produce DAG and inositol 145-trisphosphate (IP3) Diacylglycerol and IP3 are second
messengers that can activate the protein kinase C and trigger Ca2+
release respectively
The role of PLA may be regulating the composition of plasma membrane lipid
composition during low temperature stress however the signaling pathway through
which AFP induction is still not clear It cannot be ruled out that the AFP signaling
occurs through the activation of PLC but further studies are needed to validate the data
Many plants respond to cold and other abiotic stress by accumulating high amount
of compatible osmolytes such as proline sugars or sugar alcohols (mannitol) glycine-
betaine etc (Hoekstra et al 2001) The transcript abundance of P5CS was higher in WT
plants as compared to the transgenic plants (both the lines) in our studies The free
proline was also quantified in the leaf samples of transgenic and WT plants before and
after cold stress This analysis revealed that proline level was increased in WT plants
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during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
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further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
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Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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United States of America 923903-3907
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tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
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induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
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Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
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temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
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2164-11-73
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of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
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MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
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signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
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39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
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the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
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characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
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sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
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components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
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Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
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tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
during stress as compared to that of the transgenic plants which resembles as that of our
sq RT- PCR data [fig516(a) 516(b)] Proline is considered as a well-known compatible
solute that protects cell membranes and proteins against the adverse effect of inorganic
ions and temperature extremes by scavenging reactive oxygen species at high
concentration It has been reported that cold acclimation studies in WT Arabidopsis
resulted in 10 fold increase in proline content (Strand et al 2003) The sq RT-PCR
results showed that the P5CS from Jatropha curcas was induced by drought and salt
stress but not under cold stress (Zhuang et al 2011) It has been reported that the proline
content was elevated in tomato constitutively expressing At CBF1 (Hsieh et al 2002)
Eucalyptus saligna expressing P5CS induced proline showed 4 times higher proline level
as compared to the WT (Dibax et al 2010) Hence it can be concluded that the AFP
expression is not expected to increase the P5CS expression in transgenic tobacco plants
and it is clear that AFP might act independent of osmolyte accumulation route in order to
protect the plant cells
Figure 516(a) 516(b) - Estimation of Free Proline in AFP Transgenic Plants
516 (a) shows the proline content in tobacco plants and 516 (b) shows the proline content in
tomato plants
516 (a) 516 (b)
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535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
242 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
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These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
244 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
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54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
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55 REFERENCES
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accumulation of active oxygen species during the hypersensitive reaction of bean
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2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
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Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
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83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
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144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
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11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
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1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
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15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
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Arabidopsis Plant Physiology 127918-927
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Physiologia Plantarum 9311-18
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18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
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19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
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13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
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22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
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plantagineum Plant Cell 12111-123
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of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
535 Analysis of Total Soluble Sugars in Transgenic Plants- In addition to enhancement of
proline biosynthesis in plants during LT stress plants also accumulate soluble sugars
which are thought to involve in protecting plant cells from damage caused by cold stress
through different routes like serving as osmoprotectants interacting with the lipid
bilayer etc In our study the amount of soluble sugars increased in WT during stress but
there was no significant change in both tobacco and tomato transgenic lines tested
[fig 517(a) 517(b)] It can be postulated that expression of AFP leads to cold tolerance
that is independent of sugar accumulation mechanism
Figure 517- Estimation of Total Soluble Sugars in Transgenic Plants with AFP
517(a) shows the sugar estimation in tobacco lines and 517(b) in tomato lines
536 Electrolytic Leakage Assay- The semi-permeability of the plasma membrane are
disrupted at low temperatures that results in leakage of electrolytes which further leads to an
increase in ionic conductivity in those tissues The degree of cold injury in plants exposed to
low temperature can be measured by the increments in tissue conductivity due to the leakage of
electrolytes and ions Membrane damage due to various stress have been evaluated through
measurements of the rates of solute and ionic leakage At controlled conditions the leakage of
WT and transgenic lines were more or less similar in the present study As the temperature was
reduced to 4degC the leakage was higher in WT plants (both tobacco and tomato) which is
517 (a) 517(b)
242 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
244 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
245 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
246 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
further increased after 48 h of stress [fig 518(a) 518(b)] The total leakage was relatively less
in WT plants as against transgenic lines clearly indicating in transgenic plants the membrane is
stabilised (as AFP prevents the ice crystal occurrence during low temperature stress) by which
it shows more tolerance In WT plants the membrane damage is responsible for the higher
leakage of electrolytes Based on the results of the phenotype (as below) and ion leakage assay
it is very clear that the expression of carrot AFP leads to the cold tolerance in transgenic tomato
and tobacco plants The phenotypic analysis under cold stress revealed that transgenic tobacco
lines were healthy when compared to the WT plants
Figure 518- Electrolytic Leakage Assay in Transgenic Tobacco and Tomato Plants
518 (a) in tobacco lines and 518 (b) in tomato lines
537 Phenotypic Analysis- Transgenic and WT tobacco seedlings of similar age were
chosen for low temperature stress treatment to study the phenotype To assess the cold
stress tolerance both transgenic and non-transformed plants were exposed to 4degC in cold
chamber grown for 3 days The experiments revealed the drastic phenotypic changes
indicating superior chilling tolerance of transgenic plants in comparison to WT plants
The growth of WT plants was retarded during the prolonged low temperature treatment
however the growth of the tobacco transgenic lines was not affected (fig 519 520) The
length of root stem number of leaves area of leaves were also affected in WT plants
518(a) 518(b)
243
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
244 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
245 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
246 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
These results indicated clearly that transgenic lines showed better cold tolerance and
enhanced recovery from the cold treatment and the WT plants did not survive A very
similar observation was reported where an insect AFP (MpAFP 149) conferred cold
tolerance in tobacco transgenic lines and performed well whereas WT plants suffered
from chlorosis wilting after exposure (Wang et al 2008)
Figure 519- Phenotypic Analysis in Transgenic Tobacco Plants
Transgenic tobacco lines 32 41 and WT plants were given cold stress at 4degC for two weeks
AFP 32 after two week
stress
AFP 32 before
stress
WT after two week
stress
WT before
stress
AFP 41 before stress
AFP 41 after two week
stress
244 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
245 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
246 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Figure 520- Phenotypic Analysis of Transgenic Tobacco Plants
Transgenic lines 32 47 and WT plants were given cold stress at 4degC for 3 weeks to determine the
effect of cold stress on morphology of plants Lower panel shows the plants which are grown at
normal conditions (which do not exhibit any phenotypic changes)
Plants after two weeks of stress
Plants after one week stress
Plants before stress
Plants after three weeks of stress
One month old plants grown at
controlled conditions
WT AFP 32 AFP 41
WT AFP 32 AFP 41
245 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
246 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
54 CONCLUSION
Improved tolerance against different stress by single gene manipulation was
reported earlier for genes involved in transcriptional activation membrane integrity and
other metabolic functions As tobacco is a model system most of the analyses were
performed only in transgenic tobacco lines In the present study molecular biochemical
physiological and phenotypic analysis did not show any induction andor change in
transgenic plants however most of the genes studied were upregulated in WT plants
The leakage of electrolytes was less in transgenic plants during cold treatment than the
WT Our results clearly demonstrate that the increased tolerance in AFP lines to cold
stress could be through the enhanced cell membrane stability It can be concluded that
AFP integration in the genome of tobacco and tomato imparted a low temperature
tolerance in transgenic plants Field studies are required to further confirm the
effectiveness of the AFP transgenic plants in the real high altitude situations
246 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
55 REFERENCES
1 Adam AL Bestwick CS Barna B amp Mansfield JW (1995) Enzymes regulating the
accumulation of active oxygen species during the hypersensitive reaction of bean
to Pseudomonas syringae pv Phaseolicola Planta 197240-249
2 Ali AA amp Alqurainy F (2006) Activities of antioxidants in plants under
environmental stress (Eds Motohashi N) pp 187-256 Transworld Research
Network India
3 Allen R (1995) Dissection of oxidative stress tolerance using transgenic plants
Plant Physiology 1071049-1054
4 Anderson JV Chevone BI amp Hess JL (1992) Seasonal variation in the antioxidant
system of eastern white pine needles Plant Physiology l98501-508
5 Atici O amp Nalbantoglu B (2003) Antifreeze proteins in higher plants
Phytochemistry 641187-1196
6 Bates LS Waldren RP amp Teare ID (1973) Rapid determination of free proline for
water-stress studies Plant and Soil 39205-207
7 Bieleskl RL (1982) Sugar alcohols In Encyclopedia of Plant Physiology
(Ed Loewus FA amp Tanner W) pp 158-192 Springer-Verlag Berlin
8 Bowler C Montague MV amp Oxborough K (1992) Superoxide dismutase and stress
tolerance Annual Review of Plant Physiology and Plant Molecular Biology 43
83-116
9 Bridger GM Yang W Falk DE amp McKersie BD (1994) Cold acclimation increases
tolerance of activated oxygen in winter cereals Journal of Plant Physiology
144235-240
10 Chen TH amp Murata N (2002) Enhancement of tolerance of abiotic stress by
metabolic engineering of betaines and other compatible solutes Current Opinion in
Plant Biology 5250-257
11 Delauney AJ amp Verma DPS (1993) Proline biosynthesis and osmoregulation in
plants Plant Journal 4215-223
247 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
12 Dibax R Deschamps C Bespalhok-Filho JC Vieira LGE Molinari HBC
De Campos MKF amp Quoirin M (2010) Organogenesis and Agrobacterium
tumifaciens-mediated transformation of Eucalyptus saligna with P5CS gene
Biologia Plantarum 546-12
13 Doyle JJ amp Doyle JL (1990) Isolation of plant DNA from fresh tissue Focus
1213-15
14 Esterbauer H amp Grill D (1978) Seasonal variation of glutathione and glutathione
reductase in needles of Picea abies L Plant Physiology 61119-121
15 Ezaki B Katsuhara M Kawamura M amp Matsumoto H (2001) Different
mechanisms of four aluminum (Al)-resistant trangenes for Al toxicity in
Arabidopsis Plant Physiology 127918-927
16 Ezaki B Yamamoto Y amp Matsumoto H (1995) Cloning and sequencing of the
cDNAs induced by aluminium treatment and Pi starvation in cultured tobacco cells
Physiologia Plantarum 9311-18
17 Floyd RA amp Nagy I (1984) Formation of long-lived hydroxyl free-radical adducts
of proline and hydroxyproline in a Fenton reaction Biochimica et Biophysica Acta
79094-97
18 Ford CW (1984) Accumulation of low molecular weight solutes in water-stressed
tropical legumes Phytochemistry 231007-1015
19 Foyer CH amp Halliwell B (1976) The presence of gluthione and glutathione
reductase in chloroplasts A proposed role in ascorbic acid metabolism Planta
13321-25
20 Foyer CH Theodoulou FL amp Delrot S (2001) The functions of inter and
intracellular glutathione transport systems in plants Trends in Plant Science 6
486-492
21 Foyer CH Trebst A amp Noctor G (2005) Signaling and integration of defense
functions of tocopherol ascorbate and glutathione In Photoprotection
Photoinhibition Gene Regulation and Environment (eds Demmig-Adams B amp
Adams WW) pp 241-268 Kluwer Academic Publishers Dordrecht The Netherlands
248 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
22 Frank W Munnik T Kerkmann K Salamini F amp Bartels D (2000) Water deficit
triggers phospholipase D activity in the resurrection plant Craterostigma
plantagineum Plant Cell 12111-123
23 Fridovich I (1991) Molecular oxygen friend or foe In Active OxygenOxidative
Stress and Plant Metabolism (eds Pell EJ amp Steffen KL) pp 1-5 American Society
of Plant Physiologists Rockville MD
24 Fung RW Wang CY Smith DL Gross KC Tao Y amp Tian M (2006)
Characterization of alternative oxidase (AOX) gene expression in response to
methyl salicylate and methyl jasmonate pre-treatment and low temperature in
tomatoes Journal of Plant Physiology 1631049-1060
25 Griffith M Ala P Yang DSC Hon WC amp Moffat B (1992) Antifreeze protein
produced endogenously in winter rye leaves Plant Physiology 100593-596
26 Harper DB amp Harvey BMR (1978) Mechanism of paraquat tolerance in perennial
ryegrass - Role of superoxide dismutase catalase and peroxidase Plant Cell amp
Environment 1211-215
27 Hirayama T Ohto C Mizoguchi T amp Shinozaki K (1995) A gene encoding a
phosphatidylinositol-specific phospholipase C is induced by dehydration and salt
stress in Arabidopsis thaliana Proceedings of National Academy of Sciences of
United States of America 923903-3907
28 Hoekstra PA Golovina EA amp Buitink J (2001) Mechanisms of plant dessication
tolerance Trends in Plant Science 6431-438
29 Hsieh TH Lee JT Charng YY amp Chan MT (2002) Tomato plants ectopically
expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress
Plant Physiology 130618-626
30 Hua ZM Yang X amp Fromm ME (2006) Activation of the NaCl and drought-
induced RD29A and RD29B promoters by constitutively active Arabidopsis
MAPKK or MAPK proteins Plant Cell amp Environment 291761-1770
31
249 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
Hwang I Sze H amp Harper JF (2000) A calcium-dependent protein kinase can
inhibit a calmodulin-stimulated Ca2+
pump (ACA2) located in the endoplasmic
reticulum of Arabidopsis Proceedings of National Academy of Sciences of United
States of America 976224-6229
32 Ito Y Saisho D Nakazono M Tsutsmi N amp Hirai A (1997) Transcript levels of
tandem- arranged alternative oxidase genes in rice are increased by low
temperature Gene 203121-129
33 Jacob T Ritchie S Assmann SM amp Gilroy S (1999) Abscisic acid signal
transduction in guard cells is mediated by phospholipase D activity Proceedings of
National Academy of Sciences United States of America 9612192-12197
34 Jain M Ghanashyam C amp Bhattacharjee A (2010)Comprehensive expression
analysis suggests overlapping and specific roles of rice glutathione S-transferase
genes during development and stress responses BMC genomics doi1011861471-
2164-11-73
35 Janda T Szalai G Rios-Gonzalez K Veisz O amp Paldi E (2003) Comparative study
of frost tolerance and antioxidant activity in cereals Plant Science 164301-306
36 John UP Polotnianka RM Sivakumaran KA Chew O Mackin L Kuiper
MJ Talbot JP Nugent GD Mautord J Schrauf GE amp Spangenberg GC (2009) Ice
recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic
Antarctic hair grass Deschampsia antarctica E Desv Plant Cell amp Environment
32336-348
37 Jonak C Kiegerl S Ligterink W Barker PJ Huskisson NS amp Hirt H (1996) Stress
signaling in plants A mitogen-activated protein kinase pathway is activated by cold
and drought Proceedings of National Academy of Sciences of United States of
America 9311274-11279
38 Khare N Goyary D Singh NK Shah P Rathore M Anandhan S Sharma D Arif
M amp Ahmed Z (2010) Transgenic tomato cv Pusa Uphar expressing a bacterial
mannitol-1-phosphate dehydrogenase gene confers abiotic stress tolerance Plant
Cell Tissue and Organ Culture 103267-277
250 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
39 Khedr AHA Abbas MA Wahid AAA Quick WP amp Abogadallah GM (2003)
Proline induces the expression of salt stress-responsive proteins and may improve
the adaptation of Pancratium maritimum L to salt-stress Journal of Experimental
Botany 542553-2562
40 Kiegerl S Cardinale F Siligan C Gross A Baudouin E Liwosz A Eklof S Till S
Bogre L Hirt H amp Meskiene I (2000) SIMKK a mitogen-activated protein kinase
(MAPK) kinase is a specific activator of the salt stress-induced MAPK SIMK
Plant Cell 122247-2258
41 Knight CA Wen D amp Laursen RA (1995) Nonequilibrium antifreeze peptides and
the recrystallization of ice Cryobiology 3223-34
42 Knight H (2000) Calcium signaling during abiotic stress in plants International
Review of Cytology 195269-325
43 Kopka J Pical C Gray JE amp Muller-Rober B (1998) Molecular and enzymatic
characterization of three phosphoinositide-specific phospholipase C isoforms from
potato Plant Physiology 116239-250
44 Kung CC (1998) Characterization of a pea gene responsive to low temperature
Botanical Bulletin of Academia Sinica 399-15
45 Li W Li M Zhang W Welti R amp Wang X (2004) The plasma membrane-bound
phospholipase δ enhances freezing tolerance in Arabidopsis thaliana Nature
Biotechnology 22427-433
46 Madamanchi NR amp Alcher RG (1991) Metabolic bases for differences in
sensitivity of two pea cultivars to sulfur dioxide Plant Physiology 9788-93
47 Matsumura T Tabayashi N Kamagata Y Souma C amp Saruyama H (2002) Wheat
catalase expressed in transgenic rice can improve tolerance against low temperature
stress Physiologia Plantarum 116317-327
48 McCue KF amp Hanson AD (1990) Drought and salt tolerance Towards
understanding and application Trends in Biotechnology 8358-362
251 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
49 Meyer AJ Brach T Marty L Kreye S Rouhier N Jacquot JP amp Hell R (2007)
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer Plant Journal 52973-986
50 Michiels A Van den Ende W Tucker M Van Riet L amp Van Laere A (2003)
Extraction of high-quality genomic DNA from latex-containing plants Analytical
Biochemistry 31585-89
51 Mittler R Vanderauwera S Gollery M amp Van Breusegem F (2004) Reactive
oxygen gene network of plants Trends in Plant Science 9490-498
52 Moller IM Jensen PE amp Hansson A (2007) Oxidative modifications to cellular
components in plants Annual Review of Plant Biology 58459-481
53 Munnik T Irvine RF amp Musgrave A (1998) Phospholipid signaling in plants
Biochimica et Biophysica Acta 1389222-272
54 Nakano Y amp Asada K (1980) Spinach chloroplasts scavenge hydrogen peroxide on
illumination Plant and Cell Physiology 211295-1307
55 Navrot N Rouhier N Gelhaye E amp Jacquot J (2007) Reactive oxygen species
generation and antioxidant systems in plant mitochondria Physiologia plantarum
129185-195
56 Okuda T Matsuda Y Yamanaka A amp Sagisaka S (1991) Abrupt increase in the
level of hydrogen-peroxide in leaves of winter-wheat is caused by cold treatment
Plant Physiology 971265-1267
57 Palmgren MG amp Sommarin M (1989) Lysophosphatidylcholine stimulates ATP
dependent proton accumulation in isolated oat root plasma membrane vesicles
Plant Physiology 901009-1014
58 Palmgren MG Sommarin M Ulvskov P amp Larsson C (1990) Effect of detergents
on the H+-ATPase activity of inside-out and right-side-out plant plasma membrane
vesicles Biochimica et Biophysica Acta 1021133-140
59 Pei ZM Ward JM Harper JF amp Schroeder JI (1996) A novel chloride channel in
Vicia faba guard cell vacuoles activated by the serinethreonine kinase CDPK
The EMBO Journal 156564-6574
252 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
60 Peroni LA Ferreira RR Figueira A Machado MA amp Machado DR (2007)
Expression profile of oxidative and antioxidative stress enzymes based on ESTs
approach of citrus Genetics and Molecular Biology 30872-880
61 Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and
tolerance in developing maize seedlings changes in antioxidant system oxidation
of proteins and lipids and protease activities The Plant Journal 101017-1026
62 Prasad TK Anderson MD Martin BA amp Stewart CR (1994) Evidence for chilling-
induced oxidative stress in maize seedlings and a regulatory role for hydrogen
peroxide Plant Cell 665-74
63 Richards KD Schott EJ Sharma YK Davis KR amp Gardner RC (1998) Aluminum
induces oxidative stress genes in Arabidopsis thaliana Plant Physiology 116
409-418
64 Roxas V Smith R Allen E amp Allen R (1997) Overexpression of glutathione
S-transferaseglutathione peroxidase enhances the growth of transgenic tobacco
seedlings during stress Nature Biotechnology 15988-991
65 Ruelland E Cantrel C Gawer M Kader JC amp Zachowski A (2002) Activation of
phospholipases C and D is an early response to a cold exposure in Arabidopsis
suspension cells Plant Physiology 130999-1007
66 Saijo Y Hata S Kyozuka J Shimamoto K amp Izui K (2000) Over-expression of a
single Ca2+
-dependent protein kinase confers both cold and saltdrought tolerance
on rice plants Plant Journal 23319-327
67 Sanders D Brownlee C amp Harper JF (1999) Communicating with calcium Plant
Cell 11691-706
68 Scebba F Sebastiani L amp Vitagliano C (1999) Protective enzymes against activated
oxygen species in wheat (Triticum aestivum L) seedlings responses to cold
acclimation Journal of Plant Physiology 155762-768
69 Shen B Jensen RG amp Bohnert HJ (1997) Increased resistance to oxidative stress in
transgenic plants by targeting mannitol biosynthesis to chloroplasts Plant
Physiology 1131177-1183
253 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
70 Shinozaki K amp Yamaguchi-Shinozaki K (2000) Molecular responses to
dehydration and low temperature differences and cross talk between two stress
signaling pathways Current Opinion in Plant Biology3217-223
71 Sidebottom C Buckley S Pudney P Twigg S Jarman C Holt C Telford J
McArthur A Worrall D Hubbard R amp Lillford P (2000) Heat-stable antifreeze
protein from grass Nature 406256
72 Singh K Foley RC amp Onate-Sanchez L (2002) Transcription factors in plant
defense and stress responses Current Opinion in Plant Biology 5430-436
73 Smallwood M Worrall D Byass L Elias L Ashford D Doucet CJ Holt C Telofrd
J Lilliford P amp Bowles DJ (1999) Isolation and characterization of a novel
antifreeze protein from carrot (Daucus carota) Biochemical Journal 340385-391
74 Strand A Foyer CH Gustafsson P Gardestrom P amp Hurry V (2003) Altering flux
through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana
modifies photosynthetic acclimation at low temperatures and the development of
freezing tolerance Plant Cell amp Environment 26523-535
75 Sugie A Naydenov N Mizuno N Nakamura C amp Takumi S (2006) Overexpression of
wheat alternative oxidase gene WAOX1a alters respiration capacity and response to
reactive oxygen species under low temperature in transgenic Arabidopsis Genes amp
Genetic Systems 81349-354
76 Takahashi S amp Murata N (2005) Interruption of the Calvin cycle inhibits the repair of
photosystem II from photodamage Biochimica et Biophysica Acta1708352-361
77 Takahashi Y amp Nagata T (1992) parB An auxin-regulated gene encoding
glutathione-S-transferase Proceedings of the National Academy of Sciences of
United States of America 8956-59
78 Takumi S Tomioka M Eto K Naydenov N amp Nakamura C (2002)
Characterization of two non-homologous nuclear genes encoding mitochondrial
alternative oxidase in common wheat Genes amp Genetic Systems 7781-88
254 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
79 Thomashow MF (1999) Plant cold acclimation Freezing tolerance genes and
regulatory mechanisms Annual Review of Plant Physiology and Plant Molecular
Biology 50571-599
80 Urao T Katagiri T Mizoguchi T Yamaguchi-Shinozaki K Hayashida N amp
Shinozaki K (1994) Two genes that encode Ca2+
-dependent protein kinases are
induced by drought and high salt stresses in Arabidopsis thaliana Molecular and
General Genetics 224331-340
81 Vanlerberghe GC amp Mc Intosh L (1992) Lower growth temperature increases
alternative pathway capacity and alternative oxidase protein in tobacco Plant
Physiology 100115-119
82 Vierheller TL amp Smith IK (1990) Effect of chilling on glutathione reductase and total
glutathione in soybean leaves (Glycine max L) Merr In Sulfur Nutrition and Sulfur
Assimilation in Higher Plants (eds Rennenberg H Brunold C de Kok LJ Stulen I)
pp 261ndash265 SPB Academic Publishers The Hague The Netherlands
83 Wang CY (1995) Temperature preconditioning affects glutathione content and
glutathione reductase activity in chilled zucchini squash Journal of Plant
Physiology 145148-152
84 Wang Y Qiu L Dai C Wang J Luo J Zhang F amp Ma J (2008) Expression of
insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers
the cold tolerance to transgenic tobacco Plant Cell Reports 271349-1358
85 Wang Y Wisniewski M Meilan R Cui M Webb R amp Fuchigami L (2005) Over
expression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling
and salt stress Journal of American Society of Horticultural Science 130167-173
86 Willekens H Chamnongpol S Davey M Schraudner M Langebartels C Van
Montagu M Inze D amp Van Camp W (1997) Catalase is a sink for H2O2 and is
indispensable for stress defence in C-3 plants The EMBO Journal 164806-4816
87 Xing Yu Jia W amp Zhang J (2007) AtMEK1 mediates stress-induced gene
expression of CAT1 catalase by triggering H2O2production in Arabidopsis Journal
of Experimental Botany 582969-2981
255 Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark
88 Xiong L amp Ishitani M (2006) Stress signal transduction components pathways
and network integration In Abiotic stress tolerance in plants toward the
improvement of global environment and food (eds Rai AK amp Takabe T) pp 3ndash29
Springer Dordercht The Netherlands
89 Xu W Liu M Shen X amp Lu C (2005) Expression of a carrot 36 kD antifreeze
protein gene improves cold stress tolerance in transgenic Tobacco Forestry Studies
in China 77-15
90 Yancey PH Clark ME Hand SC Bowlus PD amp Somero GN (1982) Living with
water stress- Evolution of osmolyte systems Science 2171214-1217
91 Yang KY Liu Y amp Zhang S (2001) Activation of a mitogen-activated protein
kinase pathway is involved in disease resistance in tobacco Proceedings of
National Academy of Sciences of United States of America 98741-746
92 Yoshida S amp Uemura M (1990) Responses of the plasma membrane to cold
acclimation and freezing stress In The Plant Plasma Membrane (eds Larsson C amp
Moller IM) pp 304ndash319 Springer-Verlag Berlin
93 Zhang JS Li CJ Wei J amp Kirkham MB (1995) Protoplasmic factors antioxidants
responses and chilling resistance in maize Plant Physiology and Biochemistry 33
567-575
94 Zhang SH Wei Y Liu JL Yu HM Yin JH Pan HY amp Baldwin TC (2011)
An apoplastic chitinase CpCHT1 isolated from the corolla of winter sweet exhibits
both antifreeze and antifungal activities Biologia Plantarum 55141-148
95 Zhang S amp Klessig DF (2001) MAPK cascades in plant defense signaling Trends
in Science 6520-527
96 Zhuang GQ Li B Guo HY Liu JL amp Chen F (2011) Molecular cloning and
characterization of P5CS gene from Jatropha curcas L African Journal of
Biotechnology 1014803-14811
256
Please purchase PDF Split-Merge on wwwverypdfcom to remove this watermark