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CHAPTER II
REVIEW OF LITERATURE
Based on the objectives of the present study, literature
survey was done for the evaluation of photosynthetic capabilities as
determined by the levels of the chlorophyll pigments involved in the
process, osmoprotectants, antioxidant status and potentials of the plants
under NaCl stress, use of ‘omic’ technologies for identification of the
proteins that are differentially expressed under salinity treatment, in
control and treated plants, role of such proteins in salinity stress
response and tolerance.
Salinity is not the alone reason which diminish the quality of the
crops, there are major forms or other issues which intensify the salt
stress in environment which can be natural or anthropogenic. Naturally
it occurs when breakdown of rocks takes place which contains high
content of calcium, chlorine, sodium, Magnesium and sulphate.
Anthropogenic activities which includes industry, agriculture,
transportation, mining, construction, deforestation and habitations has a
great impact in increasing the salinity of the soil which direct influence
the vegetative growth of the plants. It interrupts the ecological balance of
the soil (Munns, 2005; Garg and Manchanda, 2009).
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The dissolved form of increased salt concentration, when given to
the germinating seeds of the mung bean, shows a drastic effect on the
physiology, growth, development and yield, its quality. Wahid (2004)
reported that salt stress causes chlorosis, necrosis and reduced the
chlorophyll content in the plant.
The time of exposure to salinity and the severity of the salt
treatment determine the physiological and molecular changes. A high
salt concentration in soil induces changes predominantly associated
morphology; however, a low salt treatment may not result in
morphological changes but can definitely have changes in physiological
mechanisms. Plant responses to salinity occur as a rapid, osmotic phase
that inhibits growth of young leaves and another slower, ionic phase that
accelerates senescence of mature leaves.
Salinity effect on germination
Seed germination is one of the most basic and essential phases in
the growth cycle of plants that decide plant establishment and the yield
of the crops. The accessible literature revealed the effects of salinity on
the seed germination of various crops like Oryza sativa (Xu et al., 2011),
Triticum aestivum (Akbarimoghaddam et al., 2011), Zea mays (Carpıcı et
al., 2009; Khodarahmpour et al., 2012), Brassica spp. (Ibrar et al., 2003;
Ulfat et al., 2007), Glycine max (Essa 2002), Vigna spp., (Jabeen et al.,
2003) and Helianthus annuus (Mutlu and Buzcuk, 2007).
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It is well documented that salt stress has negative correlation with
seed germination and vigor (Rehman et al., 2000). Higher level of salt
stress hinders the germination of seeds while lower level of salinity
tempts a state of dormancy. Salinity have many-fold effects on the
germination process: it modifies the imbibitions of water by seeds due to
lower osmotic potential of germination media (Khan and Weber, 2008) ,
causes toxicity which changes the activity of enzymes of nucleic acid
metabolism (Gomes-Filho et al., 2008), alters protein metabolism
(Yupsanis et al., 1994; Dantas et al., 2007), upsets hormonal balance
(Khan and Rizvi, 1994), and reduces the utilization of seed reserves
(Promila and Kumar, 2000; Othman et al., 2006). It may also
unconstructively affect the ultrastructure of cell, tissue and organs
(Koyro, 2002; Rasheed, 2009).
However, there are various internal (plant) and external
(environmental) factors that affect seed germination under saline
conditions which includes nature of seed coat, seed dormancy, seed age,
seed polymorphism, seedling vigor, temperature, light, water and gasses
(Wahid et al., 2011). The germination rates and percentage of germinated
seeds at a particular time varies considerably among species and
cultivars. Lauchli and Grattan (2007) proposed a generalized relationship
between percent germination and time after adding water at different salt
levels.
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Plant Growth under salinity stress
The presence of salt in the soil reduces the water uptake capacity
of the plant, and this causes quick reduction in the growth rate. This
first stage of the growth response is due to the osmotic effect of the soil
solution containing salt, and creates a package of effects similar to water
stress (Munns, 2002 b). The means by which salinity affects growth of a
plant depend on the time scale over which the plant is exposed to salt.
Munns (2002b) recapitulated the sequential proceedings in a plant grown
in saline environment. He stated that “In the first few seconds or
minutes, water is lost from cells and shrinked. Over hours, cells recover
their original volume but the elongation rates are still reduced which led
to lower growth rates of leaf and root. Over days, cell division rates are
also affected, and contribute to lower rates of leaf and root growth. Over
weeks, changes in vegetative development and over months, changes in
reproductive development can be seen”. In plants, where Na+ and Cl–
build up in the transpiring leaves over a long period of time, resulting in
high salt concentration and leaf death. Leaf injury and death are
attributed to the high salt load in the leaf that exceeds the capacity of
salt compartmentation in the vacuoles, causing salt to build up in the
cytoplasm to toxic levels (Munns 2002a, 2005; Munns et al., 2006).
There are abundant literature indicating that plants are
particularly susceptible to salinity during the seedling and early
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vegetative growth stage. It was observed a remarkable reduction in plant
height and tiller number and leaf area index in O. sativa plants grown in
saline soil (Hasanuzzaman et al., 2009).
Under saline condition, some crops are most sensitive during
vegetative and early reproductive stages, less sensitive during flowering
and least sensitive during the seed filling stage. In all these studies, seed
weight is the yield component of interest but similar conclusions
regarding growth stage sensitivity were obtained with both determinate
crops (the grain crops) and indeterminate (cowpea) crops (Lauchli and
Grattan,2007). Salinity increased the number of sterile florets and
viability of pollen, becoming more pronounced with increased salinity.
Seed set was reduced by 38% when female plants were grown in as low
as 10mM NaCl.
In Suaeda salsa, plant height, number of branches, length of
branches and diameter of shoot were significantly affected by salt stress
which was due to the increased content of Na+ and Cl– (Guan et al.,
2011).
While studying with Glycine max, Dolatabadian et al. (2011)
observed that salinity stress considerably decreased shoot and root
weight, total biomass, plant height and leaf number. However, leaf area
was not affected by salinity stress.
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Photosynthetic pigments
One of the most prominent effects of salt stress is the shift of
photosynthetic pigment biosynthesis (Maxwell and Johnson, 2000). The
decline in Chl content under salt stress is a commonly reported
occurrence and in diverse studies and the Chl concentration were used
as a sensitive marker of the cellular metabolic state
(Chutipaijit et al., 2011). In Oryza sativa leaves, the diminution of Chl a
and b contents of leaves was observed after NaCl treatment (200mM
NaCl, 14 days) where reduction of the Chl b content of leaves (41%) was
affected more than the Chl a content (33%) (Amirjani, 2011). In another
study, Oryza sativa exposed to 100mM NaCl showed 30%, 45% and 36%
reduction in Chl a , Chl b and carotenoids (Car) contents as contrast to
control (Chutipaijit et al., 2011 ) .
Saha et al. (2010) observed a linear decrease in the levels of total
Chl, Chl a, Chl b, Car and xanthophylls as well as the intensity of Chl
fluorescence in Vigna radiata under increasing concentrations of NaCl
treatments. Contrast to control, the pigment contents decreased on an
average, by 31% for total Chl, 22% for Chl a, 45% for Chl b, 14% for
carotene and 19% for xanthophylls (Saha et al., 2010). Related with the
decline in pigment levels, there was an average 16% loss of the intensity
of Chl fluorescence as well.
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Markers for salinity stress
Plant adaptations to salinity may be related to osmotic stress
tolerance related to Na+ exclusion and tolerance of tissue to accumulated
Na+ and Cl−. Osmotic tolerance and tissue tolerance both increase the
ability to maintain growth under the influence of Na+ in the leaf tissue.
Increased osmotic tolerance is evident mainly by the increased ability to
produce newer leaves whereas tissue tolerance is evident mainly by
survival of older leaves. Membrane disorganization, reactive oxygen
species, metabolic toxicity, inhibition of photosynthesis and attenuated
nutrient acquisition are the startling factors that initiate more
catastrophic events under the influence of salt stress.
Amides and amino acids such as proline, asparagine and
aminobutyric acid, can play an important role in the osmotic adjustment
of the plant under saline conditions. Increased sugars in plants generally
serve mainly as source of carbon and energy, osmotica, stress
protectants and signal molecules while increased levels of polyphenols
under salinity stress shows the induction of secondary metabolism as
one of the defense mechanisms adapted by the plants to face saline
environment.
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Glycine betaine and proline contents in the root and shoot are seen
high under stress conditions. By measuring the concentrations of proline
and glycine betaine it can be known whether seeds are able to survive
high salt or not (Misra and Gupta, 2006). Enzymes like γ-glutamyl kinase
and Pyrroline-5-carboxylate reductase are responsible for the synthesis
of f glycine betaine and proline, while the enzyme which facilitates the
conversion of proline to glutamate is proline oxidase which reduces the
level of proline. Under salinity decreases proline oxidase increases the
proline levels (Misra and Gupta, 2006).
The accretion of osmolytes such as proline (Pro) is a distinguished
adaptive mechanism in plants against salt stress conditions. It has also
been suggested that Pro accumulation can serve as a selection criterion
for the tolerance of most species to stressed conditions (Parida and Das
2005; Ashraf and Foolad, 2007; Ahmad et al., 2009).
Since the first report on Pro buildup in wilting perennial rye grass
(Kemble and MacPherson, 1954), a number of research works has been
carried out with reference to the role of Pro as a compatible osmolyte and
osmoprotectant and its roles in salt stress tolerance. Several studies
have ascribed an antioxidant feature to Pro, suggesting ROS scavenging
activity and Pro acting as a singlet oxygen quencher (Smirnoff and
Cumbes, 1989; Matysik et al., 2002).
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Working with Arabidopsis mutants, Werner and Finkelstein (1995)
found that a Pro-deficient mutant, selected for its ability to germinate on
saline media, was incapable to continue growth on that media because it
could not accumulate Pro to the equivalent level of the wild type. Proline
also induces the expression of salt-stress-responsive proteins and may
improve the plant adaptation to salt-stress (Khedr et al., 2003).
Glycinebetaine (GB) is a small organic metabolite soluble in water
and non-toxic at high concentrations which can potentially play a
protective role against salt stress (Ashraf and Foolad, 2007; Chen and
Murata, 2008). The major role of GB in plants exposed to salt is probably
protecting cells by osmotic adjustment (Gadallah, 1999), protein
stabilization (Makela et al., 2000), photosynthetic apparatus protection
(Allakhverdiev et al., 2003; Cha-Um and Kirdmanee, 2010), and
reduction of ROS (Ashraf and Foolad, 2007).
Hydrogen peroxide
It is a general notion that H2O2 is a ROS and for many years and it
was viewed as the unavoidable and unwanted by-product of an aerobic
respiration. But recent studies have revealed that it has significant role
in redox signaling in regulating normal processes, together with oxidative
stress and thus it has been recognized as a ‘necessary evil for cell
signaling’ (Rhee, 2006). The function of H2O2 as a signaling molecule in
transduction of stress signals to the variation of expression profiles of
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target genes was also studied in plants (Hung et al., 2005; Hernandez et
al., 2010). The relationship between H2O2 and signaling networks has
been comprehensively documented for a number of stress responses
(Larkindale and Knight, 2002; Apel and Hirt, 2004; Cheeseman, 2007).
Hydrogen peroxide (H2O2) is produced predominantly in plant cells
during photosynthesis and photorespiration, and to a lesser extent, in
respiration processes. It is the most stable of the so–called reactive
oxygen species (ROS), and therefore plays a crucial role as a signaling
molecule in various physiological processes. Intra- and intercellular
levels of H2O2 increase during environmental stresses. Hydrogen peroxide
interacts with thiol-containing proteins and activates different signalling
pathways as well as transcription factors, which in turn regulate gene
expression and cell-cycle processes. Genetic systems control cellular
redox homeostasis and H2O2 signaling. In addition to photosynthetic and
respiratory metabolism, the extra cellular matrix (ECM) plays an
important role in the generation of H2O2, which regulates plant growth,
development, acclamatory and defense responses. Most of the knowledge
about H2O2 in plants has been obtained from obligate C3 plants. During
various environmental stresses the highest levels of H2O2 are observed in
the leaf veins.
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Malondialdehyde
When ROS level reaches above threshold, increased lipid
peroxidation takes place in both cellular and organellar membranes,
which, in turn, have an effect on normal cellular performance. Lipid
peroxidation exacerbates the oxidative stress through production of lipid-
derived radicals that themselves can react with and injure proteins and
DNA. The level of lipid peroxidation has been widely used as an indicator
of ROS mediated break to cell membranes under stressful conditions.
Increased peroxidation (degradation) of lipids has been reported in plants
growing under environmental stresses. Increase in lipid peroxidation
under these stresses parallels with increased production of ROS.
Malondialdehyde (MDA) is one of the final products of peroxidation of
unsaturated fatty acids in phospholipids and is responsible for cell
membrane damage.
Salinity stress results in too much generation of ROS. High salt
concentrations lead to overproduction of the ROS- O2•−, •OH, H2O2, and
1O2 by injury of the cellular electron transport within different
subcellular compartments such as chloroplasts and mitochondria, as
well as from induction of metabolic pathways such as photorespiration.
Salt stress can lead to stomatal closure, which reduces CO2 availability
in the leaves and slows down carbon fixation which, in turn, causes
exposure of chloroplasts to excessive excitation energy and over drop of
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photosynthetic electron transport system leading to enhanced generation
of ROS and induced oxidative stress. Low chloroplastic CO2/O2 ratio also
favors photorespiration leading to increased production of ROS such as
H2O2. Lofty CO2 mitigates the oxidative stress caused by salinity,
involving lower ROS generation and a better upholding of redox
homeostasis as an outcome of higher assimilation rates and lower
photorespiration. Salinity-induced ROS disrupt normal metabolism
through lipid peroxidation, denaturing proteins, and nucleic acids in
several plant species.
Seedlings of salt-sensitive cultivar showed a substantial increase in
the rate of O2•− production, elevated levels of H2O2, MDA, declined levels
of thiol, AsA and GSH and lower activity of antioxidant enzymes
compared to salt-tolerant seedlings (Hernández et al., 2000; Perez-Lopez
et al., 2009; Karray-Bouraoui et al., 2011).
Enzymic antioxidants
Defence mechanisms against free radical-induced oxidative stress
involve i) preventive mechanisms, ii) repair mechanisms, iii) physical
defences and iv) antioxidant defences. The plant defend against these
reactive oxygen species by induction of activities of certain antioxidative
enzymes such as catalase, peroxidase, polyphenol oxidase and
superoxide dismutase which scavenge reactive oxygen species
(Mittova et al., 2003). The non-enzymatic antioxidants viz. vitamin C,
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vitamin E, carotenoids and others in the protection against oxidative
stress were also reported (Kojo, 2004).
To control the levels of reactive oxygen species (ROS) and to protect
the cells under stress conditions, plant tissues contain certain soluble
enzymes scavenging ROS (SOD, CAT, POD and PPO), detoxifying lipid
peroxidation products (Glutathione s-transferases, phospholipid-hydro
peroxide and ascorbic peroxidase) and a network of low molecular mass
antioxidants (ascorbate, glutathione, phenolic compounds and
tocopherols).
The peroxidation of lipids is considered as the most damaging
process known to occur in every living organism. Membrane damage is
sometimes taken as a single parameter to determine the level of lipid
destruction under various stresses. Now, it has been recognized that
during LPO, products are formed from polyunsaturated precursors that
include small hydrocarbon fragments such as ketones, MDA, etc and
compounds related to them. Some of these compounds react with
thiobarbituric acid (TBA) to form coloured products called thiobarbituric
acid reactive substances (TBARS). LPO, in both cellular and organelle
membranes, takes place when above-threshold ROS levels are reached,
thereby not only directly affecting normal cellular functioning, but also
aggravating the oxidative stress through production of lipid-derived
radicals. The overall process of LPO involved three distinct stages:
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initiation, progression and termination steps. Initiation step involves
transition metal complexes, especially those of Fe and Cu. However, O2-
and H2O2 are capable of initiating the reactions but as OH- is sufficiently
reactive, the initiation of LPO in a membrane is initiated by the
abstraction of a hydrogen atom, in an unsaturated fatty acyl chain of a
polyunsaturated fatty acid (PUFA) residue, mainly by OH-.
In an aerobic environment, oxygen will add to the fatty acid at the
carbon-centered lipid radical to give rise to a ROO-Once initiated, ROO-
can further propagate the peroxidation chain reaction by abstracting a
hydrogen atom from adjacent PUFA side chains. The resulting lipid
hydroperoxide can easily decompose into several reactive species
including: lipid alkoxyl radicals, aldehydes (malonyldialdehyde), alkanes,
lipid epoxides, and alcohols. A single initiation event thus has the
potential to generate multiple peroxide molecules by a chain reaction.
The overall effects of LPO are to decrease membrane fluidity; make
it easier for phospholipids to exchange between the two halves of the
bilayer; increase the leakiness of the membrane to substances that do
not normally cross it other than through specific channels and damage
membrane proteins, inactivating receptors, enzymes, and ion channels. It
has also been noted that plants exposed to various Abiotic stresses
exhibit an increase in LPO due to the generation of ROS.
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Superoxide Dismutase (EC 1.15.1.1)
All aerobic organisms are continuously subjected to
potentially destructive ROS, including superoxide (O2−), lipid peroxides
(ROO�), H2O2 and the highly reactive hydroxyl radical (OH�). These ROS
are generated by metabolic processes and their concentrations can be
increased by environmental stimuli. To prevent ROS from damaging
cellular components, organisms have evolved multiple detoxification
mechanisms, including the synthesis of low-Mr antioxidant molecules
(e.g. L-ascorbic acid and glutathione) and various enzymes. O2− is an
abundant ROS that is formed by univalent electron transfer to O2 and
can contribute to the synthesis of OH-, thus control of this ROS is
essential (Halliwell and Gutteridge, 1989).
Superoxide dismutase (SOD) catalyzes the conversion of O2− to
H2O2. Three classes of SOD activity have been identified that differ by the
active site metal cofactors (Fe, Mn, or Cu and Zn). The primary
sequences of FeSOD and MnSOD apoproteins are related, whereas
CuZnSOD is distinct. Fungi and animals have CuZnSOD and MnSOD,
whereas some plants and bacteria have been demonstrated to contain all
three forms. The plant SOD isoenzymes also differ in their subcellular
location. Typically, MnSOD is mitochondrial, FeSOD is plastidic, and
CuZnSOD can be plastidic or cytosolic (Bowler et al., 1992). There are
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also reports of peroxisomal and extracellular SODs (Streller and Wingsle,
1994; Bueno et al., 1995).
The enzyme is present in all aerobic organisms and in all
subcellular components susceptible of oxidative stress (Bowler et al.,
1992). Three types of this enzyme classified by their metal cofactor, can
be found in living organisms and they are the structurally similar FeSOD
(chloroplast stroma) and MnSOD and the structurally unrelated Cu/Zn
SOD (Cytosolic and Chloroplast enzyme) (Kim et al., 2005).
The research evidences indicates a vital role for SOD in preventing
ROS-generated cell damage and death in aerobically growing organisms.
SOD is also thought to be important in converting O2− to H2O2 during the
pathogen-induced oxidative burst in animal phagocytic immune cells and
in plant cells (Desikan et al., 1996; Babior et al., 1997).
In plants exposed to photo inhibitory light, ozone, or other
environmental conditions that cause oxidative stress can increase O2−
levels (Yruela et al., 1996; Runeckles and Vaartnou, 1997). SOD plays an
essential role in attenuating plant oxidative stress in these situations. To
date the protective role of SOD in plants has been explored by transgenic
approaches, primarily through over expression or by correlation of SOD
expression to the degree of oxidative stress resistance
(Bowler et al., 1994; Alscher et al., 1997; Scandalios, 1997).
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Plants have their own enzymatic resources, such as the
metalloenzymes superoxide dismutase (SOD) and polyphenol oxidase
(PPO) to prevent oxidative damages, and both enzymes have been related
with quality loss in foods of plant origin during the ripening and post-
harvest of fruits and vegetables (Donnelly and Robinson, 1991).
Enhanced formation of ROS under stress conditions induces both
protective responses and cellular damage. The Scavanging of O2 is
achieved through an unstream enzyme, SOD which catalyses the
dismutation of superoxide dismutase to H2O2 (Bowler et al., 1992).
Cu-Zn SOD is the most abundant and the most widely distributed
in the cell. Despite current knowledge on this enzyme, it is still in doubt
whether its real catalytic activity is the same as that described in vitro.
The sequence of reactions in which it takes part would mean in vivo
eliminating a weak oxidizer and forming a strong one, requiring the
involvement of a second enzyme, a catalase, for the neutralization.
(Donnelly and Robinson, 1991).
Peroxidase (EC 1.11.1.7)
Peroxidases (POD) are widely distributed in plants and have
suggested playing roles in growth development and lignification (Mishra
et al., 1995). The intracellular level of H2O2 is regulated by a wide range
of enzymes, the most important being and peroxidases (Willekens et al.,
1995). Peroxidases are involved in the utilization of H2O2 (Hydrogen
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peroxide), which are synthesized in plant tissues by transfer of electrons
to oxygen molecules by oxidative enzymes (Padhy et al., 2000).
Peroxidases are involved in several metabolic plant processes such
as catabolising auxins, forming bridges between cell wall components
and oxidizing the cinnamyl alcohols prior to their polymerization during
formation of suberin and lignin (Lejaa et al., 2003). They appear to be
present in all parts of the living cell in response to stress and take part in
different biochemical functions in higher plants. Peroxidases polymerize
protein and lignin or suberin precursor into plant cell wall. The free
radical intermediates produced by peroxidase oxidative activity are toxic
to pathogens (Sutherland, 1991).
The induction of plant peroxidases appears to be an early event in
plant-microbes interactions. Peroxidases are haem-containing proteins
that catalyse the reduction of hydro peroxidases, especially hydrogen
peroxide to water. Higher plants possess a number of different
isoenzymes and at least 12 distinguishable isoenzymes that fall into
three sub-groups have been characterized from tobacco: the anionic (pI
3.5-4.0), moderately anionic (pI 4.5-6.5) and the cationic (pI 8.1-11)
isoenzymes. Peroxidase isoenzyme expression is tissue specific
developmentally regulated and influenced by environmental factors
(Harrison et al., 1995). Each group is thought to serve a different
function in the cell. The moderately anionic peroxidases are localized in
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the cell walls, only have moderate activity towards lignin precursors. The
cationic isoenzymes efficiently catalyze the synthesis of H2O2 from NADH
and H2O and have been localized to the central vacuole.
Peroxidases are involved in several plant defense responses
including lignification and wound healing as well as in the production of
antimicrobial radicals (Kobayashi et al., 1994). Peroxidases are also
involved in other processes, such as the inactivation of host and
pathogen enzymes by oxidized phenolics (Matern and Kneusel, 1988).
Polyphenol Oxidase (EC 1.14.18.1)
Polyphenol oxidase (PPO) is a widely distributed enzyme in plant
kingdom, which has been studied thoroughly for defining its role in
higher plants. It is responsible for hydroxylation of polyphenols, which
are responsible for inducing disease resistance in plants. PPO converts a
variety of phenolic substrates to dark-coloured polyphenols. The role of
the Polyphenol oxidases not only because of their contribution to flavor
but also for their antioxidant activities are of great interest. This enzyme
can play a significant role in overcoming various types of injuries in plant
(Moosavi et al., 2009). Polyphenolics are synthesized by numerous plants
as secondary metabolites. The beneficial effects from fruits and
vegetables have been assigned to natural antioxidants such as
anthocyanins and polyphenolics (Kaur and Kapoor, 2001).
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Polyphenolic compounds are reported to have diverse biological
effects, including antioxidant activity, antimutagenic, antitumor and
antibacterial characteristics (Shui and Leong, 2004). Polyphenols play an
important role in preventing oxidation of not only foods but also
biomolecules in human body. Phenolic compounds have a strong
antioxidant capacity in quenching free radicals by donating hydrogen to
reactive free radicals. Enzymatic browning is the most common defect
appearing in horticultural products (Shewfelt and Porvis, 1995) which is
caused mainly by Polyphenol oxidase (PPO) in vegetables. PPO is also a
plastid copper-enzyme and its physiological activity is quite controversial.
Its involvement in the plant’s defense mechanisms against processes
leading to traumatism is well known. However, neither its intracellular
location nor its sequence of activity peaks is congruent with a single and
specific activity of protection against external damage. Some studies
relate this enzyme with reactions associated to photosynthesis,
respiration and synthesis of phenolic compounds (Robb, 1984).
PPO is related with the redox balance of the vegetable and the
protection of the chloroplast pigments against oxidative species. During
fruit growth the amount and activity of these enzymes depend on the
stress level. Later, during processing of fruits, cell integrity will be lost,
which may structurally affect the copper-enzymes such as SOD and PPO.
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Catalase (EC 1.11.1.6)
The intracellular level of H2O2 is regulated by a wide range of
enzymes, the most important being Catalase (Willekens et al., 1995).
Catalase functions through an intermediate Catalase-H2O2 complex
(compound I) and produces water and dioxygen or can decay to the
inactive compound II. In the presence of an appropriate substrate
compound I drive the peroxidatic reaction. Compound I is a much more
effective oxidant than H2O2 itself, thus the reaction of compound I with
another H2O2 molecule represents a one-electron transfer, which splits
peroxide and produces another strong oxidant, the hydroxyl radical
(Elstner and Osswald, 1994).
Proteomics in understanding abiotic stress in plants
Proteomics has proved to be a valuable tool to identify proteins
involved in abiotic stress responses in plants, allowing functional genome
analysis. Proteins have been identified as common mechanisms of
response to various abiotic stress factors (water and salt), which are
expressed in different parts of the cell, such as enzymes related to the
removal of ROS and protein heat shock (HSP), HSP70 is particularly
common in water and salt stress. Specific response mechanisms to
stress have also been identified, such as the expression of proteins
involved in the synthesis of osmolytes, aquaporins and LEA proteins
related to water stress.
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Therefore, this omic approach contributes to the understanding of
the complex mechanisms of plant response to environmental factors.
Comparative proteomic studies on different tissues, organs, organelles
and membranes, using different biological models (mutant or transgenic
plants) allow monitoring of protein expression during different times,
contributing significantly to the understanding of the mechanisms of
adaptation of plants under certain stress conditions.
Proteomics of saline stress
Several studies related to the comparative analysis of proteome
between plants subjected to salt stress and control treatments have been
performed in species like rice (O.sativa) (Abbasi and Komatsu, 2004; Yan
et al., 2005), wheat (Triticum sp.) (Huo et al., 2004), sorghum (Sectarian
italics L.) (Veeranagamallaiah et al., 2008) and A. thaliana (Ndimba,
2005), using the techniques of 2-DE and mass spectrometry, MALDI-
TOF/TOF-MS. The results show changes in expression of proteins related
to the response to salt stress, 1,100 proteins were detected in rice,
including 34 over-regulated proteins and 20 sub-regulated, while 175
proteins were detected in sorghum, most of them overregulated
(Veeranagamallaiah et al., 2008).
Developments in proteomic technology concerning protein
separation and detection, as well as mass spectrometry-based protein
identification, have an growing impact on the study of plant responses to
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salinity stress (Parker et al., 2006; Qureshi et al., 2007; Caruso et al.,
2008). New insights have been obtained into salinity stress responses by
comparative proteome studies of salt-stressed roots from Arabidopsis
and rice (Oryza sativa). The detection of novel protein candidates
associated with salinity stress (Yan et al., 2005; Jiang et al., 2007),
discovery of alterations in protein phosphorylation patterns (Chitteti and
Peng, 2007) and, recently, the location of a salinity stress-responsive
protein to the rice root apoplast with a putative function in stress
signalling (Zhang et al., 2009) indicate the importance of ion uptake and
transport and regulation of water status and signal transduction
processes in the root.
Salt stress causes changes in more than 1,100 proteins of the
proteome of roots in rice var. Nipponbore. Twelve different proteins have
been identified using the methodology of peptide fingerprint identification
by MS and searching databases (Yan et al., 2005). Three of these
proteins were identified as enolase, four of them were previously
confirmed as proteins in salt stress response, and the remaining six were
new proteins involved in regulating metabolism energy, nitrogen and
carbon in the removal of ROS and the stability of the cytoskeleton. This
study gave further signs of the responses to salinity in rice roots and
showed the extent of the proteomic approach in studies of stress in
plants (Yan et al., 2005).
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In the proteome analysis of mutant plants in tolerant (RH8706-49)
and susceptible (H8706-34) to salinity wheat, five proteins located in the
chloroplast, were identified as an ATPase transporter H+, a glutamine
synthetic 2, a precursor protein (33kDa) involved in photosystem II and
Rubisco(1.3-bisphosphate carboxylase/oxygenase) (Huo et al., 2004).
These proteins probably play a crucial role in maintaining
chloroplast function in plants under salt stress in leaves and roots of
three cultivars of rice of the subspecies indica, Nipponbare, IR36 and
Pokkali (Abbasi and Komatsu, 2004). Eight proteins demonstrated over-
regulation in leaves in response to 50 mM NaCl for 24 h. These proteins
were identified as LSY081, LSY262 and LSY363, while five proteins were
identified as fructose bisphosphate aldose, a protein complex of PS II, a
protein 2 (OEE2) (oxygen-evolving enhancer protein 2) and superoxide
dismutase (SOD). The latter enzyme has been reported in response to
drought, low temperatures, salinity and ABA, whereas the expression of
LSY081, LSY363 and OEE2 is increased by salt stress and ABA. LSY262
was expressed in leaves and roots, the aldose bisphosphatase and two
proteins related to PS II were expressed in leaf veins and sheath. LSY363
expressed in veins, but was not detected in the leaf sheath and not in the
root. These results indicate that specific proteins are expressed in
specific organs of rice plants, suggesting a coordinated response to salt
stress (Abbasi and Komatsu, 2004).
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Furthermore, comparative proteome analysis of sorghum under
different sodium chloride concentrations (100, 150 and 200 Mm) and
control treatments showed temporal changes in total protein profile
(Veeranagamallaiah et al., 2008). The results showed 175 reproducible
and detectable protein spots. Through MS analysis, 29 different proteins
were identified that are expressed in response to salt stress, involved in
various processes such as photosynthesis (31.0%), nitrogen metabolism
(13.8%) lipid metabolism (6.8%), carbohydrate metabolism (6.8%),
nucleotide metabolism (6.8%), cell wall biogenesis (6.8%) and signal
transduction (10.3%), stress-related proteins (10.3%) and proteins with
unknown functions (7.4%). The first group represented by proteins of
photosynthesis (31%) and nitrogen metabolism (13.8%), includes
enzymes such as cytochrome P450 71D, phytochrome 1, proteins
associated with photosystem I (PS I), chloroplast precursor (PSI.EB and
EC 1.14.99) and ATP synthesis among others. In the second group,
enzymes include those related to nitrogen metabolism such as glutamine
synthetase, an isoform at the roots of sorghum (EC 6.3.1.2), urease (EC
3.5.1.5) and glutamate synthesis dependent ferredoxin (EC 1.4.7.1) (FD-
GOGAT) (Veeranagamallaiah et al., 2008)
Developments in proteomic technologies may aid researchers in
understanding the plant engineering involved in important processes like
nutrition, crop yields, and defense. Proteomics applications and
advancements in technology such as multidimensional protein
36
fractionation, isobaric tags for relative and absolute quantitation, label-
free quantification mass spectrometry, and phosphoprotein and
glycoprotein enrichment and tagging, will enable the discovery of proteins
and novel regulatory mechanisms that occur during salt stress signaling
and related metabolic pathways.
The integration of proteomics with transcriptomics, metabolomics,
and bioinformatics, has facilitated insights into the molecular networks
underlying salt stress response and tolerance. These data will be used to
analyze how different components interact, and generate responses to
control divergent metabolic pathways. The proteomics community needs
to work in concert with those working in transcriptomics, metabolomics,
and bioinformatics, to reveal the mechanisms affecting plant growth and
the stress response.