11

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).

12

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).

13

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.

14

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

15

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.

16

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.

17

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.

18

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).

19

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

20

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.

21

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

22

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,

23

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:

24

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.

25

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

26

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).

27

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

28

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

29

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).

30

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.

31

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.

32

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

33

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).

34

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).

35

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.