Research Article Physiological and Biochemical Changes in...

Research ArticlePhysiological and Biochemical Changes in Brassica junceaPlants under Cd-Induced Stress

Dhriti Kapoor Satwinderjeet Kaur and Renu Bhardwaj

Department of Botanical and Environmental Sciences Guru Nanak Dev University Amritsar Punjab 143005 India

Correspondence should be addressed to Renu Bhardwaj renubhardwaj82gmailcom

Received 30 April 2014 Revised 30 May 2014 Accepted 17 June 2014 Published 15 July 2014

Academic Editor Gang Liu

Copyright copy 2014 Dhriti Kapoor et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Plants of Brassica juncea L var RLC-1 were exposed for 30 days to different concentrations (0 02 04 and 06mM) of cadmium(Cd) to analyze the Cd uptake H

2O2content hormonal profiling level of photosynthetic pigments (chlorophyll carotenoid

and flavonoid) gaseous exchange parameters (photosynthetic rate vapour pressure deficit intercellular CO2concentration

and intrinsic mesophyll rate) antioxidative enzymes (superoxide dismutase polyphenol oxidase glutathione-S transferase andglutathione peroxidase) antioxidant assays (DPPH ABTS and total phenolic content) and polyphenols Results of the presentstudy revealed the increasedH

2O2content andCd uptakewith increasingmetal doses UPLC analysis of plants showed the presence

of various polyphenols Gaseous exchangemeasurements were done by infrared gas analyzer (IRGA) which was negatively affectedby metal treatment In addition LCMS study showed the variation in the expression of plant hormones Level of photosyntheticpigments and activities of antioxidative enzymes were altered significantly in response to metal treatment In conclusion theantioxidative defence system of plants got activated due to heavy metal stress which protects the plants by scavenging free radicals

1 Introduction

Heavy metals are chief environmental pollutants and theirescalating toxicity causes threat for ecological and environ-mental reasons [1] The principle cause of the prolongedpresence of heavy metals in the environment is their non-biodegradable nature [2] When come in contact with thesoil surface they get fervently adsorbed followed by gradualadsorption and distribution in the soil Plants exposed tothese metals tend to accumulate them which immenselyaffect their growth and development These can not be eitherdegraded or transformed into harmless compounds via anybiological processes Due to this they can persist in theenvironment for long durations [3] Over a period of timethey enter and accumulate in the human body through foodchain which may further cause various health effects that areirreversible in nature [4]

The primary response of plants due to heavy metalsstress is the production of reactive oxygen species (ROS)ROS are the partially reduced forms of atmospheric oxygenand their production is strongly regulated in normal growth

conditions Consequences of heavy metal toxicity elicit theoxidative stress in plants [5] Phytotoxicity caused by heavymetals lead to stunted growth leaf chlorosis and veinnecrosis and negatively affects the development of roots andleaves and also fruit quality and quantity [6 7] [8] Cd isenormously toxic metal which causes reduction in stomataldensity and CO

2conductance and also alters the metabolic

processes like respiration and nutritional status of plantsTheenhanced level of ROS due to Cd stress triggers damage toDNA and leads to mutation [4]

In response to the stress plants possess various protectivemechanisms like chelation detoxification exclusion of metalions through phytoremediation and activation of variousstress protective proteins and osmolytes and so forth [9ndash11]Brassica juncea L is an amphiploid species which belongs toBrassicaceae family It is an oilseed crop mainly grown as afood crop and also used for its medicinal purposes It is oneof the richest sources of iron vitamin A and vitamin C andalso contains potassium calcium riboflavin thiamine and 120573-carotene It has antiseptic diuretic emetic and rubefacientproperties It has been reported to contain antioxidants like

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 726070 13 pageshttpdxdoiorg1011552014726070

2 BioMed Research International

flavonoids carotenes lutein indoles and zeaxanthin [12]The present work was undertaken to study the effects of Cdon metal uptake H

2O2content hormonal profiling level

of photosynthetic pigments gaseous exchange parametersantioxidative enzymes antioxidant assays and polyphenolsin Brassica juncea plants

2 Materials and Methods

To study the effects of Cd metal on Brassica juncea plantsa field experiment was conducted in Botanical Garden ofGuru Nanak Dev University Amritsar India 20 times 20 feetarea was taken for the experimentation and soil manure ina ratio of 3 1 was added to it The certified and disease-freeseeds of Brassica juncea L var RLC-1 were procured fromPunjab Agricultural University Ludhiana Punjab Indiaand surface-sterilized with 001mercuric chloride solutionfollowed by the repeated washing of sterile double distilledwater (DDW) Seeds were sown in different blocks Differenttreatments of Cd metal were given (0 02 04 and 06mMCd) Plants were then harvested after 30 days of germinationto study following parameters

21 Cadmium Accumulation Dried plant samples were firstdigested by the method given by Allen et al [13] 05 gof dried plant samples was taken in digested by nitricacid perchloric acid (2 1) Digested samples were cooledfiltered and diluted up to 50mL by DDW The heavymetal measurement was performed with atomic absorptionspectrophotometer (Shimadzu 6200) The metal content wasdetermined by calibration with standard curve made withdifferent concentrations of metals

22 H2O2Content H

2O2content was measured by the

method given by Velikova et al [14] To 500mg of plantmate-rial 2mL of TCA was added and centrifuged at 12000 rpmfor 15 minutes Then 05mL of 10mM PPB and 1mL of1M potassium iodide was added to 05mL of supernatantAbsorbance was taken at 390 nm Concentrations of H

2O2

were calculated against the standard curve

23 LCMS Analysis of Plant Hormones Plant samples weresubjected to LCMS in order to identify the presence of planthormones like brassinosteroids polyamines auxins abscisicacid jasmonic acid salicylic acid and gibberellic acid

Sample Preparation 5 g of fresh plant sample was homoge-nized in 40mL of 80 methanol Mixture was vortexed andcentrifuged 02mL of mixture was diluted to 4mL with 80methanol and filtered by filter papers of 022 micron poresize 2 120583L of sample was injected for LCMS study Total runtime of sample required in positive mode was 16 minutesand 6 minutes in negative mode Agilent 1100 LC has beencoupled with Bruker make mass spectrometer model Esquire3000 PDA detector was used in the instrument for detectingcompounds Temperature of column was 40∘C The solventsystem includes solvent A (water with 05 formic acid) andsolvent B (methanol)

24 Photosynthetic Pigments

241 Chlorophyll Content Chlorophyll content was mea-sured by following the method given by Arnon [15] 1 g freshplant tissue was homogenized by using 4mL of 80 acetoneThe homogenized material was subjected to centrifugationusing Eltek cooling centrifuge for 20 minutes at 13000 rpmat a temperature of 4∘CThe supernatant of plant extract wasused for the analysis of chlorophyll content The absorbanceof the supernatant was taken at 645 and 663 nm

Calculations Consider

Total Chlorophyll Content

= (Absorbance645times 202) + (Absorbance

663times 83)

times (V1000timesW)

Chlorophyll A content

= Absorbance663times (0058) minus (Absorbance

645) times 0032

Chlorophyll B content

= Absorbance645times (0096)

minus (Absorbance663) times 001872

(1)

242 Total Carotenoid Content Carotenoid content wasestimated by Maclachlan and Zalik [16] method 1 g freshplant tissue was homogenized by using 4mL of 80 acetoneThe crushed material was subjected to centrifugation usingEltek cooling centrifuge for 20minutes at 13000 rpm at a tem-perature of 4∘C The supernatant from the plant extract wasused for the analysis of chlorophyll content The absorbanceof the supernatant was taken at 480 and 510 nm


Total carotenoid content

= 76 (OD480) minus 149 (OD

510) times (

VdtimesW times 1000)

(2)

243 Total Flavonoid Content Total flavonoid content wasestimated by the method given by Kim et al [17]

Preparation of Extract 1 g of fresh plant tissue was homog-enized in chilled pestle and mortar using 3mL of absolutemethanol The crushed material was then subjected to cen-trifugation using Eltek cooling centrifuge for 20 minutes at13000 rpm at a temperature of 4∘C The supernatant fromthe plant extract was collected for the further analysis of totalflavonoid content 1mL of the plant extract was added to 4mLof double distilled water 03mL of sodium nitrite (NaNO

2)

and 03mL of aluminum chloride (AlCl3) were added to

it Then incubation was given for 5 minutes Followed byaddition of 2mL sodium hydroxide (NaOH) pink color was

BioMed Research International 3

developedThen 24mL of distilled water was added to it andabsorbance was taken at 510 nm 1mgmL of rutin was usedas standard for flavonoid content determination

25 Gaseous Exchange Parameters Gaseous exchangeparameters of plants like photosynthetic rate vapourpressure deficit intercellular CO

2concentration and

mesophyll intrinsic rate were measured with the helpof infrared gas analyzer (IRGA) (Li-COR 6400) Themeasurement was performed within the time period 900ndash1100 hmaintaining the air temperature air relative humidityCO2concentration and photosynthetic photon flux density

(PPFD) at 25∘C 80ndash90 400 120583molmolminus1 and 1000120583molmminus2sminus1 respectively

26 Antioxidative Enzymes

Preparation of Extract 1 g of harvested plant material wascrushed in prechilled pestle and mortar using 3mL of100mM potassium phosphate buffer (PPB) having pH 70The crushed material was then subjected to centrifugationusing Eltek cooling centrifuge for 20minutes at 13000 rpm at4∘C The supernatant from leaf extract was collected for thevarious biochemical analyses

261 Superoxide Dismutase (SOD) Activity Superoxide dis-mutase was estimated according to method given by Kono[18] The method is based on the principle of the inhibitoryeffect of SOD on the reduction of nitroblue tetrazolium(NBT) dye by superoxide radicals which are generatedby the autooxidation of hydroxylamine hydrochloride Thereaction mixture containing 13mL sodium carbonate buffer500120583L NBT and 100 120583L Triton X-100 was taken in the testcuvettes The reaction was initiated by the addition of 100 120583Lhydroxylamine hydrochloride After 2 minutes 70 120583L of theenzyme extract was added The percent inhibition at the rateof NBT reduction was recorded as increase in absorbance at540 nm

262 Polyphenol Oxidase (PPO) Activity Activity of PPOwas estimated according to the method given by Kumar andKhan [19] Polyphenol oxidase catalyses the o-hydroxylationof monophenol (catechol) to o-diphenols and further catal-yses the oxidation of o-diphenols to produce o-quinones(benzoquinones) 225mL of reaction mixture contained1mL PPB 05mL catechol and 025mL of enzyme sampleand then the reactionwas held for 2minutes at 25∘CReactionwas accomplished by adding 05mL of 25N H

2SO4 The

absorbance was read at 495 nm

263 Glutathione-S Transferase (GST) Activity Activity ofGST was measured according to the method described byHabig et al [20] Glutathione-S-transferase catalyzes thereaction of pharmacologically active compounds with ndashSHgroup of reduced glutathione (GSH) thereby neutralizingtheir electrophilic sites rendering the product more watersoluble The reaction was carried out in a total reactionmixture of 225mL containing 2mL PPB (02M) pH 74

100 120583L GSH (20mM) 100 120583 CDNB (20mM) and 50 120583Lenzyme sample The change in absorbance at 340 nm wasrecorded

264 Glutathione Peroxidase (GPOX) Activity GPOX activ-ity was analyzed according to the method of Flohe andGunzler [21] GPOX stimulates the production of GSSG fromGSH and H

2O2 GR causes reduction of GSSH and NADPH

oxidation is measured at 340 nm In 1mL of reaction mixture500120583L PPB 100 120583L EDTA 100 120583LNADPH and 100 120583LH

2O2

were added to a test tube Then 50 120583L of enzyme extractwas added to it Decrease in absorbance due to oxidation ofNADPH was measured after 1 minute

27 Antioxidant Assays

Preparation of Extract Plant samples (20mg) were washedand oven-dried and extracted with 80 methanol for 24hours Extract was then filtered with Whatman number1 filter paper Supernatant was used for performing thefollowing assays in ELISA reader (Biotek Synergy HT)

271 DPPH Radical Scavenging Activity This assay was per-formed according to the method given by Blois [22] 01mMDPPH was mixed in plant extract The absorbance was takenat 517 nmafter 20minutes of incubation at room temperature

Calculations The inhibitory percentage of DPPH was calcu-lated according to the following equation

Inhibition = Ac minus AsActimes 100 (3)

272 ABTS Radical Scavenging Assay ABTS radical scav-enging assay was performed by Re et al [23] method Mixed221015840-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) andpotassium persulphate in 1 05 was left for 16 hours It wasdiluted with ethanol to bring the absorbance to 07 nm Thissolution was added to the supernatant and absorbance wastaken at 734 nm

Calculations The inhibitory percentage of ABTS was calcu-lated according to the following equation


273 Total Phenolic Content Total phenolic content wasdetermined according to a procedure described by Singletonand Rossi [24] In 04 g of dried plant material 40mL of60 ethanol was added Shaking in water was done at 60∘Cfor 10min Extract was then filtered and diluted to 100mLwith 60 ethanol From diluted plant sample 25mL wastaken and redilutedwith 25mLof distilledwater 2mL samplewas mixed with 10mL of FC reagent and then after 5min2mL of 75 sodium carbonate solution was added to thereaction mixture 2 h incubation was given to the mixtureThe absorbance readings were taken at 765 nm Gallic acidwas used as a reference standard


+ESI scan 1 (232ndash240min 36 scans) frag = 1350V 2Dd

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650 700

Counts versus mass-to-charge (mz)28-HBL-4932

49320

times102

(a)

+ESI scan 1 (38ndash44min 27 scans) frag = 1350V 01d252

151

050

60 70 80 90 100 110 120 130 140 150 160

Put-889

8890

Counts versus mass-to-charge (mz)

times103

(b)

+ESI scan 1 (173ndash201min 126 scans) frag = 1350V 01d

2

15

1

05

0280 320 360 400 440 480 520 560

Typhasterol-446244620

Collision energy Ionization modeFragmentor voltage135 0 ESI


times103

(c)

Figure 1 Hormonal profiling of control plants of 30-day-old Brassica juncea


Papaverine-3402 dolicholide-479


times103

43210

100 150 200 250 300 350 400 450 500 550 600 650 700

34020

47900


(a)

minusESI scan 2 (15ndash24min 38 scans) frag = 1350V 01d

ABA-2648 24-EBL-4987 JA-2488


times103

3

2

1

0100 150 200 250 300 350 400 450 500

24880

26480

49870


(b)


IAA-1749 cadaverine-1041

times104

0

10410

17490

08060402

60 100 140 180 220 260 300 340 380


(c)

Figure 2 Hormonal profiling of 02mM Cd treated plants of Brassica juncea (expression of additional hormones 28-HBL putrescine andtyphasterol with respect to control)

Table 1 Effect of Cd metal on Cd uptake and H2O2 content of 30-day-old B juncea plants

Days of harvestingTreatments

Cd uptake (120583g gminus1 DW) H2O2 content (120583mol gminus1 FW)0mM 02mM 04mM 06mM 0mM 02mM 04mM 06mM

30 days 00 plusmn 00c 7876 plusmn 265b 8583 plusmn 161b 9378 plusmn 129a 44 plusmn 025b 459 plusmn 030b 593 plusmn 042a 593 plusmn 006a

Data presented in mean plusmn SE Different letters (a b amp c) within various concentrations of Cd (0 02 and 04mM) are significantly different (Fisher LSD posthoc test 119875 le 005)


Table 2 Hormonal profiling of Brassica juncea plants exposed to different concentrations of Cd

S number Treatments Hormones

1 Control Papaverine dolicholide abscisic acid (ABA) 24-epibrassinolide (EBL) jasmonic acid (JA) indole-3-acetic acid(IAA) and cadaverine

2 02mMCd Papaverine dolicholide ABA JA IAA cadaverine 28-homobrassinolide (HBL) putrescine (Put) andtyphasterol

3 04mMCd Papaverine dolicholide ABA 24-EBL JA IAA cadaverine 28-HBL Put typhasterol and gibberellic acid

4 06mMCd Papaverine dolicholide ABA 24-EBL JA IAA cadaverine 28-HBL Put typhasterol gibberellic acid andsalicylic acid

Table 3 Effect of Cd metal on total chlorophyll Chl A Chl B carotenoid and flavonoid content of 30- day-old B juncea plants

Treatments Total Chl (mg gminus1 FW) Chl A (mg gminus1 FW) Chl B (mg gminus1 FW) Carotenoid (mg gminus1 FW) Flavonoid (mg gminus1 FW)00mM 319 plusmn 111a 985 plusmn 101a 144 plusmn 056ab 1231 plusmn 031a 1041 plusmn 071a

02mM 2709 plusmn 114ab 736 plusmn 071ab 1273 plusmn 071a 1112 plusmn 042ab 669 plusmn 038b

04mM 2421 plusmn 131bc 408 plusmn 059b 1239 plusmn 070a 1028 plusmn 032bc 558 plusmn 034b

06mM 1972 plusmn 070c 495 plusmn 152b 1386 plusmn 014b 915 plusmn 023c 482 plusmn 019b


Gibberellic acid-3468

6

4

2

060 100 140 180 220 260 300 340

times103


34680

Figure 3 Hormonal profiling of 04mM Cd treated plants ofBrassica juncea (expression of additional hormone gibberellic acidwith respect to other treatments)


Salicylic acid-1389

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650

times103


13890

Figure 4 Hormonal profiling of 06mM Cd treated plants ofBrassica juncea (expression of additional hormone salicylic acidwith respect to other treatments)

28 UPLC Analysis of Polyphenols

Sample Preparation 5 g of plant samples was homogenizedin 40mL of 80 methanol Centrifugation was done at13000 rpm at 4∘C temperature Then supernatant was filteredwith 022micron pore size filter paper and subjected toUPLCfor the identification of various polyphenols like gallic acid(C7H6O5) epicatechin (C

15H14O6) caffeic acid (C

9H8O4)

coumaric acid (C9H8O3) ellagic acid (C

14H6O8) quercetin

(C15H10O7) and kaempferol (C

15H10O6) and was thinned

withmethanolTheplant sampleswere analyzed by ShimadzuUPLC Nexera system (Shimadzu USA) coupled with photo-diode array detector C18 column (150mm times 46mm) witha pore size of 5120583m is used at 25∘C temperature at roomtemperature with a flow rate of 1mLmin at 120582 280 nm Thesolvent system included solvent A (001 acetic acid in water)and solvent B (methanol) Injection volume was 5120583L Peakswere determined using software provided with ShimadzuUPLC Nexera system (USA) The calibration curves weregenerated by plotting concentrations versus peak areas Thedetection of every compound was based on a combination ofretention time and spectral similarity

Statistical Analysis Each experiment was conducted in threereplicates Data was expressed in Mean plusmn SE To check thestatistical significant difference between the treatments one-way ANOVA was carried out by using Assistat version 77beta

3 Results

31 Metal Accumulation Study Significant uptake of Cdmetal was observed in B juncea plants after 30 days ofsowing (Table 1) A dose-dependent increase in uptake wasfoundwith increasing concentration of CdMaximumuptake(9378 120583g gminus1 DW) was noticed in 06mM treated plants thanin 04mM (8583 120583g gminus1DW) and 02mM (7876120583g gminus1DW)respectively Control plants did not show any metal uptake

32 H2O2Content In present study B juncea plants showed

slight changes in levels of H2O2

in Cd metal treatedplants when compared to untreated ones (Table 1) Withthe increasing dose of Cd H

2O2content was increased

in dose-dependent manner Maximum content of H2O2

(593 120583mol gminus1 FW) was noticed in 06mM Cd treatmentSimilar value of H

2O2content was recorded in 04mM and

06mM concentration of Cd Level of H2O2was found lowest

in control plants (44 120583mol gminus1 FW)


0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)

0 02 04 06Concentration (mM)

02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)

Figure 5 Cd metal effect on total chlorophyll Chl A Chl B carotenoid and flavonoid content of 30-day-old B juncea plants

33 Hormonal Profiling by LCMS Plant hormones namelypapaverine dolicholide cadaverine abscisic acid 24-epibrassinolide indole 3-acetic acid and jasmonic acidwere identified in control plants (Figure 1) Following thatplant hormones got activated with enhancing doses of Cdmetal At 02mM Cd typhasterol 28-homobrassinolideand putrescine (Figure 2) at 04mM Cd gibberellic acid(Figure 3) and at 06mMCd salicylic acid was also expressed(Figure 4 Table 2)


341 Chlorophyll Content A significant decrease in totalchlorophyll content was observed in 30-day plants (Table 3

Figure 5) 162-fold reduction in total chlorophyll content wasnoticed from control (319mg gminus1 FW) plants to 06mM Cd(1972mg gminus1 FW) Chl A content was recorded maximumin control plants (985mg gminus1 FW) whereas 04mM and06mM Cd showed a very slight variation in Chl A levelwhere 06mM Cd contained more Chl A (495mg gminus1 FW)as compared to 04mM Cd treatment (408mg gminus1 FW) Cdtreatment caused very less changes in the level of Chl BLowest content of Chl B was recorded in the plants exposedto 04mM Cd (1239mg gminus1 FW) as compared to untreatedcontrol (144mg gminus1 FW) Lowest Cd toxicity was observedin the plants treated with 06mM concentration where Chl Bcontent was highest (1386mg gminus1 FW) among all treatmentsof Cd which is followed by 02mM Cd (1239mg gminus1 FW)


0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

Figure 6 Cd metal effect on photosynthetic rate vapour pressure deficit intercellular CO2concentration and intrinsic mesophyll rate of

30-day-old B juncea Plants

342 Total Carotenoid Content B juncea plants pointedout drop in the carotenoid content with the increasingconcentration of Cd (Table 3 Figure 5) Carotenoid contentwas highest in untreated control (1231mg gminus1 FW) and itgot maximum decrease (915mg gminus1 FW) with the highestconcentration of Cd that is at 06mM Cd

343 Total Flavonoid Content Results revealed the signifi-cant decrease in flavonoid content from control (1041mg gminus1FW) to 06mM Cd (482mg gminus1 FW) 216-fold decrease inflavonoid content was noticed at 06mM Cd treatment incomparison to control 02mM and 04mM Cd treatmentshowed reduction in flavonoid level from 669 to 558mg gminus1FW respectively (Table 3 Figure 5)

35 Gaseous Exchange Parameters

351 Photosynthetic Rate Cd toxicity decreased the photo-synthetic rate in 30-day-old plants ofB juncea as compared tocontrol plants (542mmol CO

2mminus2 sminus1) (Table 4 Figure 6)

Minimumphotosynthetic rate was noted in the plants treatedwith 06mM of Cd (337mmol CO

2mminus2 sminus1) At 04mM Cd

treatment (435mmol CO2mminus2 sminus1) photosynthetic rate was

found to enhance as compared to 02mM Cd (391mmolCO2mminus2 sminus1)

352 Vapour Pressure Deficit Cd metal toxicity altered thelevel of vapour pressure deficit Vapour pressure deficitdecreased with increasing Cd metal concentration (Table 4Figure 6) Highest value was recorded in the control plants(045 kPa) which decreased at 02mM Cd stressed plants(043 kPa) At 04mM Cd treatment minimum vapour pres-sure deficit was observed (034 kPa) which is lower than06mM Cd treatment (04 kPa)

353 Intercellular CO2Concentration (Ci) A continuous

decline was noticed in the intercellular CO2concentration

when Cd treatment was given to plants (Table 4 Figure 6)Lowest value was observed in 06mM Cd stressed plants(41237 ppm) Decrease in Ci value was recorded from control(42707 ppm) to 04mM Cd (41744 ppm)

354 Intrinsic Mesophyll Rate Very small variation wasnoticed in intrinsic mesophyll rate Maximum value waspossessed by control plants (0012mmol CO

2mminus3) With

metal treatment highest mesophyll rate was recorded in04mM Cd treatment (0011mmol CO

2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)

Figure 7 Cd metal effect on activities of SOD PPO GST and GPOX of 30-day-old B juncea plants

0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)

Figure 8 Cd metal effect on scavenging activities of DPPH ABTS and total phenolic content of 30-day-old B juncea plants


Table 4 Effect of Cd metal on photosynthetic rate vapour pressure deficit intercellular CO2 concentration and intrinsic mesophyll rate of30-day-old B juncea plants

Treatments Photosynthetic rate(mmol CO2 m

minus2sminus1)

Vapour pressuredeficit(kPa)

Intercellular CO2concentration

(ppm)

Intrinsic mesophyllrate

(mmol CO2 mminus3)

00mM 542 plusmn 024a 045 plusmn 0005a 42707 plusmn 072a 0012 plusmn 0001a

02mM 391 plusmn 037b 043 plusmn 0006a 42248 plusmn 168ab 0009 plusmn 0001ab

04mM 435 plusmn 021ab 034 plusmn 0004c 41744 plusmn 167bc 0011 plusmn 0001ab

06mM 337 plusmn 03b 04 plusmn 0003b 41237 plusmn 126c 0008 plusmn 00005b

Table 5 Effect of Cd metal on specific activities of SOD PPO GST and GPOX of 30-day-old B juncea plants

Treatments SOD (UAmgminus1 protein) PPO (UAmgminus1 protein) GST (UAmgminus1 protein) GPOX (UAmgminus1 protein)00mM 312 plusmn 007ab 444 plusmn 044ab 608 plusmn 009c 91 plusmn 023b

02mM 402 plusmn 022ab 619 plusmn 108ab 763 plusmn 037bc 1152 plusmn 077b

04mM 395 plusmn 035b 403 plusmn 111b 883 plusmn 037ab 1549 plusmn 037a


slightly lower than control 02mM (0009mmol CO2mminus3)

and 06mM Cd (0008mmol CO2mminus3) stress showed nearly

similar intrinsic mesophyll rate (Table 4 Figure 6)

36 Antioxidative Enzymes Activities of all the enzymesSOD PPO GST and GPOX were enhanced with theincreased dose of Cd compared to control plants (Table 5Figure 7) A continuous increase in the activity of GST wasobserved Minimum activity of enzyme was measured incontrol plants that is 608UAmgminus1 protein Cd toxicityenhanced the activity of GST from 02mM (763UAmgminus1protein) to 06mM Cd (969UAmgminus1 protein) Highestmetal treatment showed highest activity of enzyme Resultsrevealed the maximum GPOX activity at 04mM Cd treatedplants as compared to untreated control (91 UAmgminus1 pro-tein) Activity of GPOX enzyme at 02 and 06mM Cd was1152 and 1483UAmgminus1 protein respectively Slight varia-tions in activities of SOD and PPO enzymes were noticedin present study Untreated control plants showed the lowestenzymes activities (312 and 444UAmgminus1 protein resp)Then got increase in the activities from control to 02mMCd stressed plants An increase in SOD activity from 312to 402UAmgminus1 protein and from 444 to 619UAmgminus1protein for PPO was observed Activities of enzymes wereagain inhibited at 04mM Cd treated plants At 04mM Cdtreatment activities of SOD and PPO decreased to 395 and403UAmgminus1 protein respectively in comparison to 02mMCd Further 06mM Cd toxicity caused rise in enzymeactivities from 312 to 395UAmgminus1 protein (SOD) and from444 to 403UAmgminus1 protein (PPO)


371 DPPH Results revealed the increase in scavenging ofDPPH radical by Cd metal treated plants in comparison tocontrol (6069) DPPH inhibition was enhanced maximumat 06mM stressed plants (7655) In 02mM Cd and

04mM Cd stressed plants inhibition of DPPH radical wasobserved (6466 and 7202 resp) (Table 6 Figure 8)

372 ABTS In present study 06mM Cd (7355) wasfound to possess maximum potential to scavenge ABTS ascompared to control (6411) (Table 6 Figure 8) Very lessdifference in scavenging potential was observed between04mM (7346) and 06mm Cd treatment (7355)

373 Total Phenolic Content With increasing Cd toxicitytotal phenolic content also increased in dose-dependentmanner (Table 6 Figure 8) Phenol content was found max-imum in 06mM Cd stressed plants that is 1059mg gminus1FW in comparison to control plants (826mg gminus1 FW) Anincrease was also observed from 826 to 961 (02mM) and99mg gminus1 FW (04mM Cd)

38 UPLC Analysis of Polyphenols Chromatograph showedthat gallic acid caffeic acid coumaric acid ellagic acidquercetin and kaempferol were identified in the presentstudy (Figure 9 Table 7) In 02mM Cd stress ellagic acidquercetin and kaempferol were expressed and one additionalpolyphenol namely epicatechin was also observed in com-parison to control (Figure 10) Distinct peaks of quercetinand kaempferol showed their more expression in 04mMand 06mM Cd stressed plants as compared to untreatedcontrol (Figures 11 and 12 resp) Percentage of the phenoliccompounds is given in Table 6

4 Discussion

Heavy metal stress has become a foremost focal pointdue to the increased environmental pollution Metals arenonbiodegradable so they often cause lethal biological effects[25] Heavy metals lead to the formation of oxidantsfreeradicals It is the primary response of plants exposed tostress Reduced forms of atmospheric oxygen (O

2) are the


Table 6 Effect of Cd metal on scavenging activities of DPPH ABTS and total phenolic content of 30- day-old B juncea plants

Treatments DPPH () ABTS () Total phenolic content (mg gminus1 FW)00mM 6069 plusmn 142b 6411 plusmn 157b 826 plusmn 076ab

02mM 6466 plusmn 259ab 6544 plusmn 315ab 961 plusmn 049a

04mM 7202 plusmn 417ab 7346 plusmn 308a 99 plusmn 031a

06mM 7655 plusmn 184a 7355 plusmn 229a 1059 plusmn 047b

Table 7 Concentrations of phenolic compounds of 30-day-old Brassica juncea plants treated with Cd stress

S number Polyphenolic compound PercentageControl 02mMCd 04mMCd 06mMCd

1 Gallic acid 0743 mdash mdash mdash2 Caffeic acid 0143 mdash mdash mdash3 Coumaric acid 0138 mdash mdash 00844 Ellagic acid 5868 2914 2510 20455 Quercetin 0437 0473 0468 09486 Kaempferol 1561 0487 6285 47007 Epicatechin mdash 0338 mdash mdash

intermediates of ROS Generation of ROS results from theexcitation of O

2 which forms the singlet oxygen (1O

2)

These intermediates are formed from the transfer of electronswhich generate hydrogen peroxide (H

2O2) superoxide rad-

ical (O2

∙minus) and hydroxyl radical (HO∙minus) [26] Present studyalso showed the increased level of H

2O2with increasing Cd

doses It may be due to the destabilization of membrane inplants with increasing metal stress [27] as the plants werefound to accumulate more Cd with enhancing its dosesProduction of ROS occurs due to oxidative stress or throughHaber-Weiss reactions [5] Various deleterious effects of freeradicals collectively cause oxidative stress Serious imbalanceis caused in antioxidative system due to the production ofreactive oxygen species (ROS) and reactive nitrogen species(RNS) during oxidative stress

Plants possess certain stress protective mechanisms suchas antioxidative defence systems which include plant growthregulators and antioxidative enzymes [28] Antioxidativeenzymes like SOD POD PPO and GPOX help in thescavenging of free radicals Certain stress protective proteinslike heat shock proteins protect plants against oxidativedamage [29] Due to heavy metal toxicity several types ofdefence responses are produced in plants but their actiondepend upon the doses type of plant species and so forth[30] Ability of plants to ameliorate the heavymetal toxicity orto bear the stressmakes them survive in those conditions [31]Exposure of heavy metals activates the antioxidative defencesystem Similarly in the present work increased activities ofSOD PPO GST and GPOX enzymes were stimulated withmetal treatment and thus helped in the scavenging of freeradicals like DPPH These results are in coherence with thefindings of Doganlar et al [32] Antioxidative potential ofplant was enhanced in dose-dependent manner

Another mechanism of defense in plants involves thesecondary metabolites and PGRs Plant hormones like aux-ins abscisic acid brassinosteroids and polyamines regulatemetabolic processes related to plant growth and development

(mAU

)15

10

5

0

151050 20 25

(min)

PDA multi 1 280nm 4nm

Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

Figure 9 UPLC chromatograph of control plants of 30-day-oldBrassica juncea

151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00

Figure 10 UPLC chromatograph of 02mM Cd treated 30-day-oldplants of Brassica juncea

and they have also been found to work as stress protectants byscavenging the reactive oxygen species [33] These hormonesactivate the antioxidative defence system of plants exposedto stress and thus help in amelioration of stress [34 35]Similarly in present study hormones were much expressedin metal treated plants These results were supported by


151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00


the findings of Groppa et al [36 37] where putrescinebiosynthesis was found to enhance under Cu and Cd stressin sunflower discs The rise in putrescine synthesis was dueto increased activities of ornithine decarboxylase (ODC)and arginine decarboxylase (ADC) enzymes which leads tosynthesis of hormone Similarly Atici et al [38] recordedsignificant rise in the endogenous levels of ABA in the seedsof chick pea exposed to Zn and Pb stress The present workwas also in coherence with the findings of Munzuroglu etal [39] where Hg Cu and Cd toxicity caused significantenhancement in the ABA in wheat seeds

Level of photosynthetic pigments was recorded todecrease in the present investigation with increasing Cddoses Similar findings were reported in tomato mustardand garden cress [40ndash42] when exposed to Cd metal It maybe due to the fact that Cd causes inhibition of Fe and leadsto chlorosis of leaves thus negatively affecting chlorophyllmetabolism [43] Micronutrients are also degraded by thetoxicity of heavy metals which are required for the growthand development of plants Consequently level of pigmentsfalls under metal stress [44] This is also one of majorreasons which lead to photosynthesis impairment Similarresults were obtained from the present work where fall ingaseous exchange measurements was observed These resultsare in coherence with the findings of Januskaitiene [45]where gaseous exchange parameters like photosynthetic rateintercellular CO

2concentration and so forth decreased with

Cd metal stress in pea plants

5 Conclusion

Cd is one of the most toxic heavy metals which increases theproduction of ROS like H

2O2 Metabolic activities are altered

by Cd stress Various defence mechanisms of Brassica junceaplants got activated to combat the stress like antioxidativedefence system and hormonal level Thus the plantsrsquo owndefensive strategies provide protection to plants from oxida-tive stress generated by Cd

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors are grateful to the University Grant Commission(UGC) for providing fellowship and also thankful to Botani-cal and Environmental Sciences GuruNanakDevUniversityAmritsar India for providing laboratory facilities for thiswork

References

[1] P C Nagajyoti K D Lee and T V M Sreekanth ldquoHeavy met-als occurrence and toxicity for plants a reviewrdquo EnvironmentalChemistry Letters vol 8 no 3 pp 199ndash216 2010

[2] R Singh N Gautam A Mishra and R Gupta ldquoHeavy metalsand living systems an overviewrdquo Indian Journal of Pharmacol-ogy vol 43 no 3 pp 246ndash253 2011

[3] R Ramasubbu and C Prabha Effect of Heavy Metals on Growthand Biochemical Components of Plants LAP Lambert AcademicPublishing 2012

[4] G Flora D Gupta and A Tiwari ldquoToxicity of lead a reviewwith recent updatesrdquo Interdisciplinary Toxicology vol 5 no 2pp 47ndash58 2012

[5] AMithofer B Schulze andW Boland ldquoBiotic and heavymetalstress response in plants evidence for common signalsrdquo FEBSLetters vol 566 no 1ndash3 pp 1ndash5 2004

[6] A Elbaz Y Y Wei Q Meng Q Zheng and Z M YangldquoMercury-induced oxidative stress and impact on antioxidantenzymes in Chlamydomonas reinhardtiirdquo Ecotoxicology vol 19no 7 pp 1285ndash1293 2010

[7] D K Meng J Chen and Z M Yang ldquoEnhancement oftolerance of Indian mustard (Brassica juncea) to mercury bycarbon monoxiderdquo Journal of Hazardous Materials vol 186 no2-3 pp 1823ndash1829 2011

[8] H Li M Jiang L L Che L Nie and Z M Yang ldquoBjHO-1 isinvolved in the detoxification of heavy metal in India mustard(Brassica juncea)rdquo BioMetals vol 25 no 6 pp 1269ndash1279 2012

[9] T Vamerali M Bandiera and G Mosca ldquoField crops forphytoremediation of metal-contaminated land A reviewrdquo Envi-ronmental Chemistry Letters vol 8 no 1 pp 1ndash17 2010

[10] L W Zhang J B Song X X Shu Y Zhang and Z M YangldquomiR395 is involved in detoxification of cadmium in Brassicanapusrdquo Journal of Hazardous Materials vol 250-251 pp 204ndash211 2013

[11] Z S Zhou S N YangH Li C C Zhu Z P Liu and ZM YangldquoMolecular dissection of mercury-responsive transcriptome


and senseantisense genes in Medicago truncatulardquo Journal ofHazardous Materials vol 252-253 pp 123ndash131 2013

[12] K Shekhawat S S Rathore O P Premi B K Kandpal andJ S Chauhan ldquoAdvances in agronomic management of indianmustard (Brassica juncea (L) Czernj Cosson) an overviewrdquoInternational Journal of Agronomy vol 2012 Article ID 40828414 pages 2012

[13] S E Allen H M Grimshaw and A P Rowland ldquoChemicalanalysisrdquo in Methods in Plant Ecology S B Chapman Ed pp311ndash314 Blackwell Scientific Publications Oxford UK 1976

[14] V Velikova I Yordanov and A Edreva ldquoOxidative stressand some antioxidant systems in acid rain-treated bean plantsprotective role of exogenous polyaminesrdquo Plant Science vol 151no 1 pp 59ndash66 2000

[15] D I Arnon ldquoCopper enzymes in isolated chloroplasts Pho-tophenoloxidase in Beta vulgarisrdquo Plant Physiology vol 24 pp1ndash15 1949

[16] S Maclachlan and S Zalik ldquoPlastid structure chlorophyllconcentration and free amino acid composition of a chlorophyllmutant of barleyrdquo Canadian Journal of Botany vol 41 no 7 pp1053ndash1062 1963

[17] M S Kim C Kim D H Jo and Y W Ryu ldquoEffect of fungalelicitor and heavy metals on the production of flavonol glyco-sides in cell cultures of Ginkgo bilobardquo Journal of Microbiologyand Biotechnology vol 9 no 5 pp 661ndash667 1999

[18] Y Kono ldquoGeneration of superoxide radical during autoxidationof hydroxylamine and an assay for superoxide dismutaserdquoArchives of Biochemistry and Biophysics vol 186 no 1 pp 189ndash195 1978

[19] K B Kumar and P A Khan ldquoPeroxidase amp polyphenol oxidasein excised ragi (Eleusine corocana cv PR 202) leaves duringsenescencerdquo Indian Journal of Experimental Biology vol 20 no5 pp 412ndash416 1982

[20] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquoThe Journal of Biological Chemistry vol 249 no 22pp 7130ndash7139 1974

[21] L Flohe and W A Gunzler ldquoAssays of glutathione peroxidaserdquoMethods in Enzymology vol 105 pp 114ndash121 1984

[22] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[23] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[24] V L Singleton and J A Rossi ldquoColorimetry of total pheno-lics with phosphomolybdicphosphotungstic acid reagentsrdquoTheAmerican Journal of Enology andViticulture vol 16 pp 144ndash1581965

[25] C A Jaleel K Jayakumar Z C Xing and M M AzoozldquoAntioxidant potentials protect Vigna radiata (L) Wilczekplants from soil cobalt stress and improve growth and pigmentcompositionrdquo Plant Omics vol 2 pp 120ndash126 2009

[26] N la Rocca C Andreoli G M Giacometti N Rascio and IMoro ldquoResponses of the antarctic microalga Koliella antarctica(Trebouxiophyceae Chlorophyta) to cadmium contaminationrdquoPhotosynthetica vol 47 no 3 pp 471ndash479 2009

[27] M Srivastava L Q Ma N Singh and S Singh ldquoAntioxidantresponses of hyper-accumulator and sensitive fern species toarsenicrdquo Journal of Experimental Botany vol 56 no 415 pp1335ndash1342 2005

[28] J Chen and Z M Yang ldquoMercury toxicity molecular responseand tolerance in higher plantsrdquoBioMetals vol 25 no 5 pp 847ndash857 2012

[29] J K Donnelly and D S Robinson ldquoSuperoxide dismutaserdquo inOxidative Enzymes in Foods D S Robinson andN AM EskinEds pp 49ndash91 Elsevier Applied Science London UK 1991

[30] A Arora R K Sairam and G C Srivastava ldquoOxidative stressand antioxidative system in plantsrdquo Current Science vol 82 no10 pp 1227ndash1238 2002

[31] J L Hall ldquoCellular mechanisms for heavy metal detoxificationand tolerancerdquo Journal of Experimental Botany vol 53 no 366pp 1ndash11 2002

[32] Z B Doganlar S Cakmak and T Yanik ldquoMetal uptake andphysiological changes in Lemna gibba exposed to manganeseand nickelrdquo International Journal of Biology vol 4 pp 148ndash1572012

[33] P Sharma and R Bhardwaj ldquoEffect of 24-epibrssinolide onseed germination seedling growth and heavy metal uptake inBrassica juncea Lrdquo General and Applied Plant Physiology vol33 no 1-2 pp 59ndash73 2007

[34] L L Haubrick and S M Assmann ldquoBrassinosteroids andplant function Some clues more puzzlesrdquo Plant Cell andEnvironment vol 29 no 3 pp 446ndash457 2006

[35] A Verma C P Malik and V K Gupta ldquoIn vitro effectsof brassinosteroids on the growth and antioxidant enzymeactivities in groundnutrdquo ISRN Agronomy vol 2012 Article ID356485 8 pages 2012

[36] M D Groppa M L Tomaro andM P Benavides ldquoPolyaminesand heavy metal stress the antioxidant behavior of spermine incadmium- and copper-treated wheat leavesrdquo BioMetals vol 20no 2 pp 185ndash195 2007

[37] M D Groppa M S Zawoznik M L Tomaro and M P Bena-vides ldquoInhibition of root growth and polyamine metabolism insunflower (Helianthus annuus) seedlings under cadmium andcopper stressrdquo Biological Trace Element Research vol 126 no1ndash3 pp 246ndash256 2008

[38] O Atici G Agar and P Battal ldquoChanges in phytohormonecontents in chickpea seeds germinating under lead or zincstressrdquo Biologia Plantarum vol 49 no 2 pp 215ndash222 2005

[39] OMunzuroglu F K Zengin andZ Yahyagil ldquoThe abscisic acidlevels of wheat (Triticum aestivum L cv cakmak 79) seeds thatwere germinated under heavymetal (Hg++ Cd++ Cu++) stressrdquoGazi University Journal of Science vol 21 no 1 pp 1ndash7 2008

[40] M Mobin and N A Khan ldquoPhotosynthetic activity pig-ment composition and antioxidative response of two mustard(Brassica juncea) cultivars differing in photosynthetic capacitysubjected to cadmium stressrdquo Journal of Plant Physiology vol164 no 5 pp 601ndash610 2007

[41] A F Lopez-Millan R Sagardoy M Solanas A Abadıaand J Abadıa ldquoCadmium toxicity in tomato (Lycopersiconesculentum) plants grown in hydroponicsrdquo Environmental andExperimental Botany vol 65 no 2-3 pp 376ndash385 2009

[42] S S Gill N A Khan and N Tuteja ldquoCadmium at highdose perturbs growth photosynthesis and nitrogenmetabolismwhile at low dose it up regulates sulfur assimilation andantioxidant machinery in garden cress (Lepidium sativum L)rdquoPlant Science vol 182 no 1 pp 112ndash120 2012

[43] C Chaffei K Pageau A Suzuki H Gouia M H Ghorbel andCMasclaux-Daubresse ldquoCadmium toxicity induced changes innitrogen management in Lycopersicon esculentum leading to ametabolic safeguard through an amino acid storage strategyrdquoPlant amp Cell Physiology vol 45 no 11 pp 1681ndash1693 2004


[44] L M Sandalio H C Dalurzo M Gomez M C Romero-Puertas and L A del Rıo ldquoCadmium-induced changes in thegrowth and oxidative metabolism of pea plantsrdquo Journal ofExperimental Botany vol 52 no 364 pp 2115ndash2126 2001

[45] I Januskaitiene ldquoImpact of low concentration of cadmium onphotosynthesis and growth of pea and barleyrdquo EnvironmentalResearch Engineering and Management vol 3 no 53 pp 24ndash29 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


flavonoids carotenes lutein indoles and zeaxanthin [12]The present work was undertaken to study the effects of Cdon metal uptake H

2O2content hormonal profiling level

of photosynthetic pigments gaseous exchange parametersantioxidative enzymes antioxidant assays and polyphenolsin Brassica juncea plants

2 Materials and Methods

To study the effects of Cd metal on Brassica juncea plantsa field experiment was conducted in Botanical Garden ofGuru Nanak Dev University Amritsar India 20 times 20 feetarea was taken for the experimentation and soil manure ina ratio of 3 1 was added to it The certified and disease-freeseeds of Brassica juncea L var RLC-1 were procured fromPunjab Agricultural University Ludhiana Punjab Indiaand surface-sterilized with 001mercuric chloride solutionfollowed by the repeated washing of sterile double distilledwater (DDW) Seeds were sown in different blocks Differenttreatments of Cd metal were given (0 02 04 and 06mMCd) Plants were then harvested after 30 days of germinationto study following parameters

21 Cadmium Accumulation Dried plant samples were firstdigested by the method given by Allen et al [13] 05 gof dried plant samples was taken in digested by nitricacid perchloric acid (2 1) Digested samples were cooledfiltered and diluted up to 50mL by DDW The heavymetal measurement was performed with atomic absorptionspectrophotometer (Shimadzu 6200) The metal content wasdetermined by calibration with standard curve made withdifferent concentrations of metals

22 H2O2Content H

2O2content was measured by the

method given by Velikova et al [14] To 500mg of plantmate-rial 2mL of TCA was added and centrifuged at 12000 rpmfor 15 minutes Then 05mL of 10mM PPB and 1mL of1M potassium iodide was added to 05mL of supernatantAbsorbance was taken at 390 nm Concentrations of H

2O2

were calculated against the standard curve

23 LCMS Analysis of Plant Hormones Plant samples weresubjected to LCMS in order to identify the presence of planthormones like brassinosteroids polyamines auxins abscisicacid jasmonic acid salicylic acid and gibberellic acid

Sample Preparation 5 g of fresh plant sample was homoge-nized in 40mL of 80 methanol Mixture was vortexed andcentrifuged 02mL of mixture was diluted to 4mL with 80methanol and filtered by filter papers of 022 micron poresize 2 120583L of sample was injected for LCMS study Total runtime of sample required in positive mode was 16 minutesand 6 minutes in negative mode Agilent 1100 LC has beencoupled with Bruker make mass spectrometer model Esquire3000 PDA detector was used in the instrument for detectingcompounds Temperature of column was 40∘C The solventsystem includes solvent A (water with 05 formic acid) andsolvent B (methanol)


241 Chlorophyll Content Chlorophyll content was mea-sured by following the method given by Arnon [15] 1 g freshplant tissue was homogenized by using 4mL of 80 acetoneThe homogenized material was subjected to centrifugationusing Eltek cooling centrifuge for 20 minutes at 13000 rpmat a temperature of 4∘CThe supernatant of plant extract wasused for the analysis of chlorophyll content The absorbanceof the supernatant was taken at 645 and 663 nm


Total Chlorophyll Content

= (Absorbance645times 202) + (Absorbance

663times 83)

times (V1000timesW)

Chlorophyll A content

= Absorbance663times (0058) minus (Absorbance

645) times 0032

Chlorophyll B content

= Absorbance645times (0096)

minus (Absorbance663) times 001872

(1)

242 Total Carotenoid Content Carotenoid content wasestimated by Maclachlan and Zalik [16] method 1 g freshplant tissue was homogenized by using 4mL of 80 acetoneThe crushed material was subjected to centrifugation usingEltek cooling centrifuge for 20minutes at 13000 rpm at a tem-perature of 4∘C The supernatant from the plant extract wasused for the analysis of chlorophyll content The absorbanceof the supernatant was taken at 480 and 510 nm


Total carotenoid content

= 76 (OD480) minus 149 (OD

510) times (

VdtimesW times 1000)

(2)

243 Total Flavonoid Content Total flavonoid content wasestimated by the method given by Kim et al [17]

Preparation of Extract 1 g of fresh plant tissue was homog-enized in chilled pestle and mortar using 3mL of absolutemethanol The crushed material was then subjected to cen-trifugation using Eltek cooling centrifuge for 20 minutes at13000 rpm at a temperature of 4∘C The supernatant fromthe plant extract was collected for the further analysis of totalflavonoid content 1mL of the plant extract was added to 4mLof double distilled water 03mL of sodium nitrite (NaNO

2)

and 03mL of aluminum chloride (AlCl3) were added to

it Then incubation was given for 5 minutes Followed byaddition of 2mL sodium hydroxide (NaOH) pink color was




2concentration and







2SO4 The







2O2













3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650 700


49320

times102

(a)


151

050

60 70 80 90 100 110 120 130 140 150 160

Put-889

8890


times103

(b)


2

15

1

05

0280 320 360 400 440 480 520 560




times103

(c)





times103

43210

100 150 200 250 300 350 400 450 500 550 600 650 700

34020

47900


(a)


ABA-2648 24-EBL-4987 JA-2488


times103

3

2

1

0100 150 200 250 300 350 400 450 500

24880

26480

49870


(b)



times104

0

10410

17490

08060402

60 100 140 180 220 260 300 340 380


(c)





















6

4

2

060 100 140 180 220 260 300 340

times103


34680



Salicylic acid-1389

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650

times103


13890





9H8O4)


14H6O8) quercetin





3 Results












0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)


02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)







0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2concentration and







2SO4 The







2O2













3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650 700


49320

times102

(a)


151

050

60 70 80 90 100 110 120 130 140 150 160

Put-889

8890


times103

(b)


2

15

1

05

0280 320 360 400 440 480 520 560




times103

(c)





times103

43210

100 150 200 250 300 350 400 450 500 550 600 650 700

34020

47900


(a)


ABA-2648 24-EBL-4987 JA-2488


times103

3

2

1

0100 150 200 250 300 350 400 450 500

24880

26480

49870


(b)



times104

0

10410

17490

08060402

60 100 140 180 220 260 300 340 380


(c)





















6

4

2

060 100 140 180 220 260 300 340

times103


34680



Salicylic acid-1389

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650

times103


13890





9H8O4)


14H6O8) quercetin





3 Results












0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)


02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)







0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650 700


49320

times102

(a)


151

050

60 70 80 90 100 110 120 130 140 150 160

Put-889

8890


times103

(b)


2

15

1

05

0280 320 360 400 440 480 520 560




times103

(c)





times103

43210

100 150 200 250 300 350 400 450 500 550 600 650 700

34020

47900


(a)


ABA-2648 24-EBL-4987 JA-2488


times103

3

2

1

0100 150 200 250 300 350 400 450 500

24880

26480

49870


(b)



times104

0

10410

17490

08060402

60 100 140 180 220 260 300 340 380


(c)





















6

4

2

060 100 140 180 220 260 300 340

times103


34680



Salicylic acid-1389

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650

times103


13890





9H8O4)


14H6O8) quercetin





3 Results












0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)


02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)







0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















6

4

2

060 100 140 180 220 260 300 340

times103


34680



Salicylic acid-1389

3

2

1

0100 150 200 250 300 350 400 450 500 550 600 650

times103


13890





9H8O4)


14H6O8) quercetin





3 Results












0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)


02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)







0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35

0 02 04 06

a

abbc

c

Concentration (mM)

Tota

l Chl

(mg g

minus1

FW)

(a)

0

2

4

6

8

10

12

0 02 04 06

a

ab

b

b

Concentration (mM)

Chl A

(mg g

minus1

FW)

(b)


02468

10121416 ab

a ab

Chl B

(mg g

minus1

FW)

(c)


0

2

4

6

8

10

12

14a

abbc

c

Caro

teno

id (m

g gminus1

FW)

(d)


0

2

4

6

8

10

12 a

bb

b

Flav

onoi

d (m

g gminus1

FW)

(e)







0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5

6

0 02 04 06

a

b ab

b

Concentration (mM)

Phot

osyn

thet

ic ra

te (m

mol

CO2 m

minus2

sminus1 )

(a)


0005

01015

02025

03035

04045

05 a a

cb

Vapo

ur p

ress

ure d

efici

t (kP

a)

(b)


400

405

410

415

420

425

430 aab

bc

c

Inte

rcel

lula

rCO

2co

ncen

trat

ion

(ppm

)

(c)


0

0002

0004

0006

0008

001

0012

0014 a

ab

ab

b

Intr

insic

mes

ophy

ll ra

te (m

g CO

2m

minus3)

(d)

















2mminus3) With


2mminus3) which was


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5

6

0 02 04 06

ab

ab b

a

Concentration (mM)

SOD

(UA

mgminus

1pr

otei

n)

(a)

0123456789

ab

ab

b

a

PPO

(UA

mgminus

1pr

otei

n)


(b)

0

2

4

6

8

10

12a

abbc

c

GST

(UA

mgminus

1pr

otei

n)


(c)

02468

1012141618

b

b

a a

GPO

X (U

A m

gminus1

prot

ein)


(d)


0102030405060708090

0 02 04 06

DPP

H (

)

bab

ab a

Concentration (mM)

(a)


0102030405060708090

ABT

S (

)

b aba a

(b)


0

2

4

6

8

10

12

aba a

b

Tota

l phe

nolic

cont

ent (

mg

FW)

gminus1

(c)





minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




minus2sminus1)



(ppm)


(mmol CO2 mminus3)




















4 Discussion


2) are the












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












2)



ical (O2






(mAU

)15

10

5

0

151050 20 25

(min)


Gal

lic ac

id

Caffe

ic ac

id

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol


151050 20 25

(min)


Epic

atec

hin

Ella

gic a

cid

Que

rcet

inKa

empf

erol

(mAU

)

50

25

00




151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


151050 20 25

(min)


(mAU

)

Ella

gic a

cid

Que

rcet

inKa

empf

erol

4

3

2

1

0


151050 20 25

(min)


(mAU

)

Cou

mar

ic ac

id

Ella

gic a

cid

Que

rcet

inKa

empf

erol

75

50

25

00






5 Conclusion






Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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