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Plants are exposed to various environmental factors (biotic as well as abiotic) which constitute their macro and microenvironment. The abiotic factors include high temperature, cold, drought, salinity, and the biotic factors are viruses, insects, nematodes, bacteria, fungi etc. Amongst these stresses, salinity has emerged as one of the most serious factors limiting productivity of agricultural crops (Allakhverdiev et al., 2000b). Salinity stress biology and plant responses to high salinity have been discussed over two decades (Flowers et al., 1977; Greenway and Munns, 1980; Ehret and Plant, 1999; Hasegawa et al., 2000; Zhu, 2002). Salinization of land has threatened civilizations in ancient and modern times. Soil salinization in southern Mesopotamia and in several parts of the Tigris-Euphrates alluvial plains of Iraq destroyed the ancient societies that had successfully thrived for several centuries (Jacobsen and Adams, 1958; Hillel, 2005; Rengasamy 2006). Salt-affected lands occur in practically all climatic regions, from the humid tropics to the polar regions. In modern times, salt-affected soils are naturally present in more than 100 countries of the world where many regions are also affected by irrigation-induced salinization.
Salt stress imposes osmotic stress on a wide variety of metabolic activities (Greenway and Munns, 1980; Cheeseman, 1988). This osmotic stress leads to secondary stress the oxidative stress. During oxidative stress the formation of reactive oxygen species (ROS) such as superoxide (O2-î º), hydrogen peroxide (H2O2), hydroxyl radical (-OH) (Halliwell and Gutteridge, 1985), and singlet oxygen (1O2) (Elstner, 1987) takes place.
ROS are free radicals that are atoms or groups of atoms having at least one unpaired electron. This is a highly unstable configuration, so the radicals promptly react with other molecules to generate more free radicals, because electrons tend to pair upto give rise stable two electron bonds (Foyer and Halliwell, 1976; Hideg, 1997). Production of ROS takes place by a number of different mechanisms (Figure 1).
Salt stress reduces gas exchange thereby limiting CO2 supply to the leaf (Asada, 1999). This causes the over-reduction of the photosynthetic electron transport chain (Asencio et al., 2003). This causes oxidative stress in plants (Foyer and Noctor, 2003; Ali and Alqurainy, 2006) thereby causing the production of ROS such as singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical (Asada,1999; Kiddle et al., 2003; Gomez et al., 2004;Mateo et al., 2004).
ROS targets are high-molecular mass molecules, such as membrane lipids or mitochondrial DNA, with the formation of lipid or nucleotide peroxides, especially at the level of thymine (Cullis et al. 1987). ROS is capable of inducing damage to almost all cellular macromolecules including DNA (Tuteja et al. 2008).
The polyunsaturated fatty acids (PUFAs) are the major fatty acids in the plant membrane and are particularly susceptible to attack by 1O2 and HO•, giving rise to complex mixtures of lipid hydroperoxides (Mueller 2004). Extensive PUFA peroxidation decreases the fluidity of the membrane, increases leakiness, and causes secondary damage to membrane proteins (Halliwell 2006). Aldehydes formed in the mitochondria may be involved in causing cytoplasmic male sterility in maize because a restorer gene in this species encodes a mitochondrial aldehyde dehydrogenase (Liu et al. 2001, Moller 2001).
DNA can be modified by ROS in many different ways. HO• is the most reactive, 1O2 primarily attacks guanine, and H2O2 and O2•− do not react at all (Wiseman and Halliwell 1996). 8-Hydroxyguanine is the most commonly observed modification.
The oxidation of sugars with HO• often releases formic acid as the main breakdown product (Isbell et al. 1973). This may be the long-sought-after source of substrate for the enigmatic enzyme, formate dehydrogenase (Igamberdievet al. 1999; Juszczuk et al. 2007).
Protein oxidation is defined here as covalent modification of a protein induced by ROS or byproducts of oxidative stress. Most types of protein oxidations are essentially irreversible, whereas, a few involving sulfur containing amino acids are reversible (Ghezzi and Bonetto 2003). Protein oxidation is widespread and often used as a diagnostic marker for oxidative stress.
The toxic effects of ROS are counteracted by enzymatic as well as non-enzymatic antioxidative system such as: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), ascorbic acid (AsA), tocopherol, glutathione and phenolic compounds etc. Normally, each cellular compartment contains more than one enzymatic activity that detoxifies a particular ROS (Fig. ). For example, the cytosol contains at least three different enzymatic activities that scavenge H2O2: APX, GPX, and PrxR (Nobuhiro and Mittler 2006). Development of genetically engineered plants by the introduction and/or overexpression of selected genes seems to be a viable option to generate abiotic stress tolerant plants (Mathur et al. 2008).
Osmotic stress induced growth arrest
Arrest of plant growth during stress conditions largely depends upon the severity of the stress. Mild osmotic stress leads rapidly to growth inhibition of leaves and stem, whereas roots may continue to elongate (Nonami and Boyer, 1990; Spollen et al., 1993). The degree of growth inhibition due to osmotic stress depends on the time scale of the response, the particular tissue and species in question and how the stress treatment was given (rapid and gradual). Growth arrest can be considered as a possibility to preserve carbohydrates for sustained metabolism, prolonged energy supply, and for better recovery after stress relief. The inhibition of shoot growth during water deficit is thought to contribute to solute accumulation and thus eventually to osmotic adjustment (Osorio et al., 1998). For instance, Hexose accumulation accounts for a large proportion of the osmotic potential in the cell elongation zone in cells of the maize root tip (Sahrp et al., 1990). On the other hand, continuation of root growth under drought stress is an adaptive mechanism that facilitates water uptake from deeper soil layers. Similarly, continued root growth under salt stress may provide additional surfaces for sequestration of toxic ions, leading to lower salt concentration. For example, salt tolerance of barley was correlated with the better root growth rates coupled with fast development and early lowering (Munns et al., 2000).
Osmotic stress affects photosynthesis (From Parida paper)
Effects of salinity on photosynthetic pigments and proteins The chlorophyll and total carotenoid contents of leaves decrease in general under salt stress. The oldest leaves start to develop chlorosis and fall with prolonged period of salt stress (Hernandez et al., 1995, 1999; Gadallah, 1999; Agastian et al., 2000). However, Wang and Nil (2000) have reported that chlorophyll content increases under conditions of salinity in Amaranthus. In Grevilea, protochlorophyll, chlorophylls, and carotenoids are significantly reduced under NaCl stress, but the rate of decline of protochlorophyll and chlorophyll is greater than that of Chl-a and carotenoids. The anthocyanin pigments on the other hand significantly increase in this case with salt stress (Kennedy and De Fillippis, 1999). In leaves of tomato, the contents of total chlorophyll (Chl-a+b), Chl-a, and b carotene decrease by NaCl stress (Khavarinejad and Mostofi, 1998). Under salinity stress, leaf pigments studied in nine genotypes of rice reduce in general, but relatively high pigment levels are found in six genotypes (Alamgir and Ali, 1999). In the cyanobacterium Spirulina platensis a decrease in the phycocyanin/chlorophyll ratio and no significant change in the carotenoid/chlorophyll ratio are observed under salt stress (Lu and Vonshak, 1999). Salinity causes significant decreases in Chl-a, Chl-b, and carotenoid in leaves of B. parviflora (Table 6; Parida et al., 2002).
Effects of salt-induced ROS on plant growth and metabolism
Although aerobic oxidative metabolism allows an efficient utilization of the energy stored in chemical bonds, the use of molecular oxygen as the final electron acceptor in the electron transport chain causes a threat of oxidative damage due to production of reactive oxygen species (Ali and Alqurainy, 2006). ROS can interact nonspecifically with various cellular components, eliciting peroxidative reactions and causing considerable damage to vital molecules such as proteins, lipids, and nucleic acids. Therefore, their levels must not exceed the optimum limit within the cells (Scandalios, 1993; Halliwell and Gutteridge, 1999; Mittler, 2002). Although a number of harmful effects of ROS on plant growth and metabolism have been widely reported in the literature, they also play an important role in many important physiological phenomena such as cell signaling, gene regulation, senescence, programmed cell death, pathogen defense, and others (Hammond-Kosack and Jones, 1996; Grant and Loake, 2000; Breusegem et al., 2001; Neill et al., 2002; Blokhina et al., 2003; Dat et al., 2000; 2003; Laloi et al., 2004; Gechev et al., 2006). It has also been observed that the presence of H2O2 in the apoplast plays a positive role, i.e., it is toxic for pathogens, plays an active role in gene transcription and systemic acquired resistance, and limits the spread of invading organisms by cell death around the infection (Horemans et al., 2000a; Smirnoff, 2000).
There is a general consensus of researchers that under normal growth conditions, the production of ROS in the cell is low and under stressful environments it is considerably high. This general statement can be confirmed by the data presented by Polle (2001) and Mittler (2002). They reported that under normal growth conditions, the production of O2 •− is as low as 240 μM s−1 and the steady state level of H2O2 in chloroplasts is 0.5 μM (Dat et al., 2000; Polle, 2001). However, in contrast, under stress conditions like salinity the production of O2•− touches the figure of 720 μM s−1 and H2O2 15 μM (Dat et al., 2000; Polle, 2001).
Superoxide is not able to react directly with proteins or lipids, but its protonated form (prehydroxyl radical) can bring about lipid peroxidation (Asada and Takahashi, 1987). Superoxide is known to inhibit peroxidases (Asada and Takahashi, 1987) and ribonucleotide reductase (Foyer and Harbison, 1994), but it causes toxicity indirectly, by accelerating the production of hydrogen peroxide and hydroxyl radicals (Hideg, 1997).
Hydrogen peroxide being the most destructive of all ROS can cause over 50% reduction in photosynthesis in plants if its concentration increases to 10 μM in the chloroplast (Kaiser, 1979). In chloroplasts, H2O2 can inhibit photosynthetic carbon assimilation by oxidizing the thiol groups of fructoe-1, 6-bisphosphatase and other important enzymes of Calvin pathway, particularly those which take part in thiol-disulfide exchange reactions (Kaiser, 1979; Tanaka et al., 1982). H2O2, in the presence of catalytic iron ions (Fe2+ or Fe3+), was reported to be very harmful as the source of hydroxyl radicals (Hideg, 1997). It may also inactivate Cu/Zn-SOD by inactivating its functional copper site (Asada et al., 1975; Bertini et al., 1989), but in contrast, it did not affect Mn containing SOD (Asada et al., 1975).
Hydroxyl radicals are not directly produced in chloroplasts; however, superoxide and hydrogen peroxide take part in a series of reactions thereby giving rise hydroxyl radicals and other destructive species such as lipid peroxides (Noctor and Foyer, 1998). Since the hydroxyl radicals have no specific scavenging enzymes in cholorplasts or in any other biological systems, so they are very reactive (Hideg, 1997). Because of being very reactive, hydroxyl radicals can bring about denaturation of proteins by oxidizing amino acids,mutation ofDNA, and peroxidation of lipids (Vaidyanathan et al., 2003).
Role of ROS in stomatal closure
Recently, some lines of studies have shown a relationship between the signal transduction pathways for abscisic acid (ABA) and the production of ROS (Jiang and Zhang 2001). Several studies revealed that ABA action on stomatal closure is mediated with the generation of ROS and cytosolic free Ca2+ (Murata et al. 2001; Pei et al. 2000). In the proposed mechanisms, ABA elicits the production of H2O2 and, in turn, the resultant H2O2 stimulates the opening of Ca2+ channels, resulting in a rapid increase in cytosolic Ca2+ concentration ([Ca2+]c). The activated Ca2+ signaling pathway triggers the closure of stomata. Similarly, fungal saccharides (especially COSs) and SA induce the closure of stomata in Commelina communis L. (Lee 1998; Lee et al. 1999), tomato (Lee et al. 1999) and Vicia faba (Mori et al. 2001) epidermis. In V. faba, SA induces a rapid production of
superoxide anion (O ·_), which is an extracellular POX dependent reaction (Mori et al. 2001). In support of this, production of ROS via tobacco POX- and horseradish peroxidase (HRP)-dependent oxidation of both SA and COSs have been demonstrated both in vivo and in vitro (Kawano et al. 2000c). However, in leaves of maize, plasma membrane (PM)-NADPH oxidase, rather than extracellularly localized POX, is reported to be involved in ABA-induced O2·_ generation and stomatal closure (Jiang and Zhang 2002). It is well recognized that plants possess both the PM-NADPH oxidase and cell-wall bound POX as two major sources for ROS production as defense mechanisms against biotic and abiotic stresses (Yoshioka et al. 2001). Mori et al. (2001) integrated our knowledge on the redox signaling networks for stomatal closure and proposed a model (Fig. 1). In the model, ABA and peptide elicitors elicit the production of ROS by stimulating the NADPH oxidase system highly sensitive to a specific inhibitor, diphenylene iodonium (DPI). On the other hand, SA and COSs induce the production of ROS via an apoplastic POX-dependent manner that is insensitive to DPI but highly sensitive to salicylhydroxamic acid (SHAM). The resultant ROS stimulates the opening of hyperpolarization-gated Ca2+-permeable channels, thus inducing an increase in [Ca2+]c in the guard cells. Consequently, downstream events associated with the PM are initiated. Such events, leading to stomatal closure, include changes in the activities of the H+-pump, anion channels, K+ channels and actin cytoskeleton. The decrease in stomatal aperture induced by elicitors and SA may be a part of the defense mechanism to limit the chances of invasion by pathogens through stomata.
Effect of Salt stress on Protoplasmic characteristic
Plasma membrane permeability
The plasma membrane is the part of the cytoplasm that first encounters the salt, and plasma membrane permeability reflects the status of the membrane lipid matrix and lipid-protein interaction. Several studies suggest that the plasma membrane may be the primary site of salt injury (Maas and Nieman, 1978; Levitt, 1980; Cramer et al., 1985; Lauchli, 1990 and Mansour, 1997). To test this hypothesis, the response of the plasma membrane to salinity in genotypes contrasting in salt tolerance was studied by measuring the plasma membrane permeability of individual cells to nonelectrolytes and water. Plasma membrane permeability probes the changes in the membrane composition and structure ( McElhaney et al., 1973; Simon, 1974; Carruthers and Melchoir, 1983; Stadelmann and Lee-Stadelmann, 1989 and Magin et al., 1990). Nonelectrolyte and water permeability was altered markedly in salt sensitive cultivars upon salt exposure ( Table 1; Mansour et al., 1993a and Mansour and Stadelmann, 1994; Mansour, 1995a and Mansour, 1995b; Mansour and Salama, 1996b). However, changes in cell membrane permeability of salt tolerant cultivars were always marginal ( Table 1). Results from other laboratories using tissue leakage as an indicator of cell membrane injury report greater leakage in salt sensitive cultivars in response to salinity ( Table 1; Poovaiah and Leopold, 1979; Dwivedi et al., 1981; Leopold and Willing, 1984 and Zongli et al., 1987).
No osmotic effect of salinity on the plasma membrane was found in several studies (Table 1). Thus, it is concluded that the deleterious effect of salinity on the plasma membrane is essentially due to the action of salt ions ( Leopold and Willing, 1984; Mansour et al., 1993a; Mansour, 1995b and Mansour, 1997). It is also interesting to mention that alterations in plasma membrane permeability have been observed in salt sensitive species without reduced growth or severe chlorosis, suggesting that membrane permeability is a sensitive test for salt stress and tolerance ( Mansour et al., 1993a and Mansour, 1997). Toxic lesions produced by salt on the membrane are expected to be more pronounced in salt sensitive plants.
Physiological data indicate that differences in cytoplasmic viscosity already exist between cells of contrasting genotypes in the absence of salt stress (Slonov, 1986; Udovenlco and Evdokimov, 1970; Mansour et al., 1993c; Mansour and Stadelmann, 1994 and Mansour and Salama, 1996b; Table 2). High cytoplasmic viscosity of salt tolerant plants was attributed to an increase in hydrophilic cytoplasmic proteins or other cytoplasmic macromolecules. These macromolecules may already be present in the cytoplasm or formed under salinity. Cytoplasmic viscosity of salt sensitive plants is not only lower than that of tolerant cultivars but also further decreases by salt imposition ( Table 2). This differential response of viscosity reflects considerable differences in cytoplasmic structure or composition between sensitive and tolerant plants. Differences in proteins that are synthesized under salinity ( LaRose et al., 1989; Hurkman et al., 1991; Plant and Bray, 1999 and Hasegawa et al., 2000) are consistent with the interpretation that variations in cytoplasmic proteins can cause alterations in cytoplasmic viscosity of contrasting cultivars. It is interesting that a number of the proteins that are induced by salinity are cytoplasmic ( Plant and Bray, 1999).
In the absence of salt imposition, however, cytoplasmic streaming was faster in the cells of a salt sensitive cultivar than in cells of salt tolerant barley seedlings (Mansour and Stadelmann, 1994; Table 2). Since cytoplasmic streaming in a plant cell is based on the functioning of the actin-myosin system requiring ATP as energy ( Mansour and Stadelmann, 1994 and references therein), a low activity of cytoplasmic streaming in salt tolerant cultivars might suggest a small cytoplasmic ATP pool or differences in other factors in the cytoplasm (e.g. free Ca2+) that can also influence streaming (Okazaki and Tazawa, 1986).
Low intensity in cytoplasmic streaming in a resistant cultivar may have physiological significance by utilizing less ATP for streaming and more ATP may, therefore, remain available for energizing metabolic processes. Furthermore, slow cytoplasmic streaming may lower the rate of metabolic transport in the cell, thus decreasing metabolic activity, resulting in slower plant growth. Reduced growth was observed in various salt tolerant plants and was interpreted to have an adaptive value in the presence of salinity (Kuiper et al., 1988; Blum, 1994 and Mansour and Salama, 1996a). It appears that slow cytoplasmic streaming may be advantageous in a saline environment. This interpretation, however, must be taken with caution until further studies with different species/varieties are carried out.
Accumulation of Sugars and compitable solutes
For the various organic osmotica, sugars contribute up to 50% of the total osmotic potential in glycophytes subject to saline conditions (Cram 1976). The accumulation of soluble carbohydrates in plants has been widely reported as a response to salinity or drought, despite a significant decrease in net CO2 assimilation rate (Murakeozy et al. 2003). Carbohydrates such as sugars (glucose, fructose, sucrose, fructans) and starch accumulate under salt stress (Parida et al., 2002). Their major functions are osmoprotection, osmotic adjustment, carbon storage, and radical scavenging. A strong correlation between sugar accumulation and osmotic stress tolerance has been widely reported, including transgenic experiments (Gilmour et al., 2000; Streeter et al., 2001; Taji e al., 2002). The increase in sugar mostly results in increased starch hydrolysis, which requires activities of hydrolytic enzymes. Sugar may protect cells during desiccation by forming glasses (Black and Pritchard, 2002). Sugars are also thought to interact with polar headgroups of phospholipids in membranes so that membrane fusion is prevented.
The sugar which has been shown to contribute to desiccation tolerance in yeast and some nematodes is trehalose (a dissccharide). It has been reported that many higher plants possess trehalose activity, which is perhaps responsible for rapid degradation of any trehalose synthesized. Arabidopsis thaliana has at least one gene which encodes trehalosse-6-phosphate phosphatase which is required for trehalose synthesis, but the physiological role of this enzyme is not clear (Vogel et al., 1998).
There is now conclusive evidence to suggest that trehalose is present in trace amounts in vascular plants, including major crops, but the actual role of this osmolyte in metabolism is still unclear. Salt stress increases reducing sugars (glucose, fructose), sucrose, and fructans in a number of plants (Kerepesi and Galiba, 2000; Khatkar and Kuhad, 2000; Singh et al., 2000). Gao et al. (1998) have reported that in tomato (Lycopersicon esculentum L.) salinity enhances sucrose concentration and activity of sucrose phosphate synthase (EC 126.96.36.199) in leaves but decreases the activity of acid invertase (EC 188.8.131.52).
Proline, which occurs widely in higher plants and in many other organisms and accumulates in larger amounts than other amino acids (Abraham et al. 2003), regulates the accumulation of useable N.
Besides osmotic adjustment other roles have been proposed for proline in osmotically stressed plant tissue: protection of plasma membrane integrity (Mansoor et al., 1998) a sink of energy or reducing power (Verbruggen et al., 1996), a source for nitrogen and carbon (Ahmad and Hellebust, 1988; Peng et al., 1996), or hydroxyl radical scavenger (Smirnoff and CUmbes, 1989; Hong et al., 2000).
Proline accumulation can occur via two biosynthetic pathways in plants: the L -ornithine and the L-glutamate pathways. It is also known that, as in plants, both ornithine and glutamate are precursors of proline biosynthesis in microorganisms and mammals. Delauney et al. (1993) showed that two enzymes: pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), play major roles in proline biosynthetic pathway (Figure------). Transgenic tobacco plants over-expressing P5CS have shown increased concentration of proline and resistance to both drought and salinity stresses (Kishor et al.1995). There are indications from Arabidopsis that the Ornithine pathway operates mainly in young seedlings (Roosens et al., 1998). The other important process that controls proline levels is oxidation of L-proline by proline dehydrogenase (ProDH) to P5C, which is converted to L-glutamic acis by P5C dehydrogenase.
Transgenic approach to improve plant stress tolerance have been reported by many workers (Roosens et al., 2002; Zhu et al., 1998). Overproduction of proline by genetically manipulated tobacco plant showed tolerance to NaCl (Hong et al. 2000). Nanjo et al. (2003) demonstrated that introduction of antisense proline dehydrogenase Cdna in Arabidopsis overexpresses proline and showed tolerance to freezing temperatures (−7°C) as well as salinity (600 mmol NaCl). The RD29A promoter- driven DREB1A transgenic plants exhibited tolerance to water deficit, salt stress, and freezing temperatures (Kasuga et al. 1999).
mRNA levels of salicylic acid-binding (SAbind) catalase (CAT) and lignin-forming peroxidase (POX) were found to be increased by proline and betaine under salt stress. It is concluded that both proline and betaine provide a protection against NaCl-induced cell death via decreasing level of ROS accumulation and lipid peroxidation as well as improvement of membrane integrity (Banu et al., 2009).
LEA proteins comprises the vast majority of stress-responsive proteins and were first identified, in cotton and wheat (Dure et al., 1981; Galau et al., 1986). LEA proteins are principally found in plant seeds and seedlings. They have also been discovered in the prokaryotes (Stacy and Aalen, 1998) as well as in the eukaryotes (Garay-Arroyo et al., 2000) such as the nematodes (Aphelenchus avenae) with functional evidences (Battista et al., 2001).
LEA proteins have been divided in to different groups based on conserved structural features (Bray et al., 1993). Group 1 LEA proteins are characterized by high glycine content (~20%), amino acid with charged R- groups (~40%) and the presence of a stretch of 20 hydrophylic amino acid. The high hydrophilicity of these proteins renders them soluble after boiling suggesting that these proteins are highly hydrated and donot assume a globular tertiary structure (Dure, 1993). The Group 1 LEA proteins may be involved in binding or replacement of water.
Group 2 proteins include lysine rich 15-amino acid sequence motif termed K-segment (EKKGIMD-KIKEKLPG) at the C-terminus and serine residues, a conserved motif containing the consensus sequence DEYGNP near the N-terminus. Group 2 proteins act as molecular chaperones and defending protein structure during stress. Four names have been designated for this protein family- RAB, LEA D-11, LEA (II) and DHNs (dehydrins) (Baker et al., 1988; Dure et al., 1989). Dehydrin (dehydration induced) genes expresses in the embryos during the late stages of embryogenesis and are also induced in vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration (e.g. drought, low temperature and salinity). They are distributed in a wide range of organisms including algae, yeast, cyanobacteria and higher plants. Dehydrins are mainly found in cytosol, nucleus mitochondria, vacuole and the vicinity of plasma membrane, (Close, 1997; Rorat, 2006).
Group 3 LEA proteins are characterised by a repeat of 11-mer amino acid motif with the consensus amino acid sequence TAQAAKEKAGE sometimes repeated as much as 13 times in Brassica napus Lea76 (Harada et al., 1989) and Hordeum vulgare HVA1. The consensus sequence could be postulated to form an alpha helical dimer suitable for the sequestration of positively and negatively charged ions that accumulate under water deficit conditions (Dure, 1993). Group 3 LEA proteins are abundant, cytosolic proteins, ubiquitous and conserved in plants. Functional analyses of LEA proteins have been carried out under both in vitro and in vivo conditions. Studies with HVA1, a barley group 3 LEA protein, conferred tolerance to water deficit and other stress conditions (Xu et al., 1996; Sivamani et al., 2000; Maqbool et al., 2002; Rohila et al., 2002). Expression of HVA1 in japonica rice (Xu et al., 1996) and Basmati rice (Rohila et al., 2002) conferred water deficit and salt stress tolerance. In transgenic rice, ion leakage due to stresses is reduced by 90 % compared to the wild type plant. The transgenic plants wherein the HVA1 gene was regulated by a stress inducible promoter, displayed less ionic leakage in comparison to plants expressing the same gene under a constitutive promoter (Rohila et al., 2002). Transgenic plants maintained higher leaf relative water content (RWC) showed lesser reduction in plant growth under drought stress, delayed wilting and better cell membrane protection (Babu et al., 2004). These results indicate that the production of HVA1 might have helped in better performance of transgenic rice plants by protecting cell membrane from injury under drought stress (Babu et al., 2004). In transgenic wheat, water use efficiency (WUE) improved by about 20 % leading to higher biomass accumulation as compared to the wild type plants (Sivamani et al., 2000). Oat transgenics showed delayed wilting under drought stress, 37 % more tolerance to salt stress and 60 % more tolerance to mannitol stress (Maqbool et al., 2002). Heterologous expression of HVA1 in yeast (Saccharomyces cerevisiae) improved the growth rate under both ionic (NaCl and KCl) as well as low temperature stresses (Zhang et al., 2000). Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry, Morus indica (Lal et al., 2008) and in Agrostis stolonifera (Fu et al., 2007).
Group 4 is characterized by a conserved N-terminal part with a random coil structure. Group 5 LEA proteins contain a higher proportion of hydrophobic residues than the other four groups and probably adopt a globular conformation. Proteins of 3 and 5 have been suggested to form dimmers with a coiled-coil structure capable of sequestering ions, which accumulate due to water depletion (Dure et al., 1989).
The polyamines (PAs) spermidine, spermine and putrescine are polycationic small aliphatic amines that are ubiquitous in all plant cells. PAs are basic molecules which are positively charged (Takeda et al., 1983) and have been shown to bind strongly in vitro to negatively charged nucleic acids (Feurstein and Marton, 1989), acidic phospholipids (Tadolini et al., 1985) and many types of proteins, including numerous enzymes whose activities are directly modulated by polyamine binding (Carley et al., 1983). These ionic interactions are important in regulating the structure and function of biological macromolecules, as well as their synthesis in vivo (Jacob and Stetler, 1989).
Differences in PA (Put, Spd, Spm) response under salt-stress have been reported among and within species. For example, according to Prakash andPrathapsenan, (1988), endogenous levels of PAs (Put, Spd and Spm) decreased in rice seedlings under NaClstress, whereas Basu et al. (1988) reported that salinity results in accumulation of these compounds in the same material. Krishnamurthy and Bhagwat, (1984) reported that salt-tolerant rice cultivars drastic cally accumulated high levels of Spd and Spm resulting in enhanced level of total PA, with a relative decrease in Put content. The most significant aspect of salt-sensitivity in rice cultivars was their excessive accumulation of Put and their inability to maintain high levels of Spd and Spm in their root systems when exposed to saline environment. In sorghum, Spd and Spm accumulation occurred during salt-stress and was considered as an adaptive response Erdei et al. (1996) Santa-Cruz et al. (1997) reported that the (Spd_Spm): Put ratios increased with salinity in the salt-tolerant tomato species (Lycopersicon pennellii, Carrel D'Arcy) but not in the salt-sensitive tomato species (L. esculentum).
A role for polyamine has been proposed in stress responses. Salt tolerant Pokkali rice plants accumulate higher levels of polyamines compared to the salt sensitive rice plants in response to salinity stress (Chattopadhyay et al., 2002). Exogenously supplied putrecine prevented stress damage and increased stress stress tolerance in Conyza bobariensis and maize (Ye et al., 1998).
Biosynthesis of polyamines in plants is controlled by the enzymes Ornithine decarboxylase (ODC; EC 184.108.40.206) and arginine decarboxylase (ADC; EC 220.127.116.11) which are responsible for the production of putrescine and S-adenosyl-L-methionine (SAM) decarboxylase that is necessary for the formation of spermidine and sepremine. Plants subjected to osmotic stress show a rapid increase in putricine levels due to transcription and activation of arginine decarboxylase (Borrel et al., 1996). Arabidopsis mutants Spe1-1 and spe2-1 with reduced activity of ADC are more sensitive to salt stress than wild type plants (Kasinathan and Wingler, 2002). Furthermore, AtADC2 expression correlated with free putrecine accumulation under salinity and dehydration (Urano et al., 2003). Transgenic rice plants with enhanced level of ADC gene expression increased biomass compared to control plants under saline conditions (Roy and Wu, 2001). Polyamines may possibly exert their protective function by scavenging ROS, which may occur as a consequence of stress (Tiburcio et al., 1994). However accumulation of polyamines seems to be toxic to the plants under normal conditions and therefore constitutive overexpression may not be the appropriate way to obtain stress tolerance.
GB is known to accumulate in response to stress in many crop plants, including sugar beet (Beta vulgaris), spinach (Spinacia oleracea), barley (Hordeum vulgare), wheat (Triticum aes- tivum), and sorghum (Sorghum bicolor) (Weimberg et al., 1984; Fallon and Phillips, 1989; McCue and Hanson, 1990; Rhodes and Hanson, 1993; Yang et al., 2003). In these species, tolerant genotypes normally accumulate more GB than sensitive genotypes in response to stress. GB is thought to protect the plant by maintaining the water balance between the plant cell and the environment and by stabilizing macromolecules (Rontein et al., 2002).
In higher plants, GB is synthesized in chloroplast from serine via ethanolamine, choline, and betaine aldehyde (Rhodes and Hanson 1993). Choline is converted to betaine aldehyde, by choline monooxygenase (CMO), which is then converted to GB by betaine aldehyde dehydrogenase (BADH) (Figure----------). Although other pathways such as direct N-methylation of glycine is also known, the pathway from choline to glycine betaine has been identified in all GB-accumulating plant species (Weretilnyk et al. 1989).
Some plant species such as rice (Oryza sativa), mustard (Brassica spp.), Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) naturally do not produce GB under stress or non-stress conditions (Rhodes and Hanson, 1993). In these species, transgenic plants with over-production of GB synthesizing genes exhibited increases in the production of GB and an enhancement in tolerance to salt, cold, drought or high temperature stress (Rhodes and Hanson, 1993). The accumulated GB in these species, however, was much lower than what is naturally found in GB-accumulating plant species under stress conditions (Rhodes and Hanson, 1993). The limitation in production of GB in high quantities in transgenic plants is reportedly due to either low availability of substrate choline or reduced transport of choline into the chloroplast where GB is naturally synthesized (Nuccio et al., 1998; Huang et al., 2000; McNeil et al., 2000).
In addition to its direct protective roles, either through positive effects on enzyme and membrane integrity or as an osmoprotectant, GB may also protect cells from environmental stresses indirectly via its role in signal transduction. For example, GB may have a role in Na+/K+ discrimination, which substantially or partially contributes to plant salt tolerance. Ion homeostasis in plants is governed by various membrane transport systems. Recently, significant progress has been made in the characterization of cation transporters that maintain ion homeostasis during salt stress in plants, of which SOS (salt overly sensitive) is a novel signaling pathway (Chinnusamy et al., 2005). This pathway is somewhat regulated by MAP kinases, expressions of which are highly affected by GB. Also, some physiological studies of GB-treated turfgrass and Arabidopsis plants indicate that GB up-regulates expression of many genes (~360 genes), of which more than 6% are known to be related to signal transduction (John, 2002). Examples are lipoxygenase, monodehydroascorbate reductase, osmotin, putative receptor kinase, calmodulin, protein kinase, and receptor protein kinase. These and other evidence have led some investigators to suggest that GB contributes to plant salt tolerance through its role in signal transduction and ion homeostasis (John, 2002; Yilmaz, 2004). However, knowledge of how GB affects expression of genes responsible for, or related to, plant salt tolerance is scarce. Elucidation of the roles of GB in regulating genes of signaling pathways used by plants to respond to environmental stresses may lead to devising approaches to improve plant stress tolerance.
Improvement of salt tolerance through engineering antioxidant genes
Undoubtedly, during the last few decades, genetic engineering approach has gained a considerable ground because progress in terms of improving a trait in a crop could be achieved within the shortest possible time period. Furthermore, tolerance to a multitude of abiotic stresses has been correlatedwellwith enhanced activities of antioxidant enzymes and levels of non-enzymaticmetabolites. Thus, the potential to engineer plants that over-express introduced antioxidant genes provides an opportunity to develop plants with enhanced tolerance to salinity.
In an attempt to produce transgenic Arabidopsis overexpressing Mn-SOD, one of the important isoforms of SOD in plant cells, Wang et al. (2004) observed that the activity of Mn-SODwas over two-fold as compared to that of wild type (Table 3). The transgenic Arabidopsis plants showed higher tolerance to salt as compared to the non- engineered plants. Further analyses revealed that despite the enhanced activities of Mn-SOD, the activities of other antioxidative enzymes such as Cu/Zn-SOD, Fe-SOD, CAT and POD of transgenic plants treated with salt were markedly higher than those of wild type plants. Also, the levels of MDA were lower in the transgenic plants than those of wild type under salt treatment, indicating the enhanced ability of the antioxidant metabolites to scavenge/detoxify ROS in transgenic Arabidopsis (Table 3). In the same model plant, enhanced expression of DHAR, an enzyme actively involved in the recycling of ascorbate by reducing the oxidized ascorbate,was achieved by the expression of rice
DHAR in transgenic Arabidopsis plants (Ushimaru et al., 2006). In fact, the transgenic Arabidopsis showed enhanced tolerance to salt despite the fact that there had been a slight increase in DHAR activity and total ascorbate in the transgenic plants. While examining the prospective role of SOD in the salt tolerance of rice using transgenic plants, Tanaka et al. (1999) reported a 1.7-fold increase in the activity of SOD in the transformed plants as compared to that in the control plants under non-saline conditions. This difference in SOD activity between transformed and non-transformed rice plants was also found under saline conditions. In addition, the ascorbate peroxidase activity of the transgenic plants was about 1.5-fold higher than that in the control plants under saline conditions. Similarly, in tobacco, Yadav et al. (2005) developed transgenic plants overexpressing glyoxalase pathway enzymes that resist an increase in accumulation of methylglyoxal (MG) in plants under salt stress. The transgenic plants showed enhanced activity of various glutathione related antioxidative enzymes under both control and saline conditions. Additionally, these plants showed less lipid peroxidation, and maintained high content of reduced glutathione and reduced to oxidized glutathione (GSH:GSSG) ratio under salt stress.
These results suggest that impediment in an increase in MG coupled with maintaining higher reduced glutathione levels can be considerably achieved by the overexpression of glyoxalase pathway enzymes for developing salt tolerant plants. In the same plant species, the role of
APX (ascorbate peroxidase) in protection against oxidative stress was investigated using transgenic plants (Badawi et al., 2004). The transgenic tobacco line Chl-APX5 showed 3.8-fold the level of APX activity as compared to that in the wild-type plants and enhanced tolerance to salt andwater stress by efficiently detoxifying hydrogen peroxide from the chloroplasts. Amaya et al. (1999) tested the germinability of seeds of transgenic tobacco plants over-expressing a cell wall peroxidase gene. They found the seeds of transgenic line highly tolerant to salt stress during germination, and suggested that this could have been due to the improved ability of transgenic seeds to retain considerable amount ofwater for germination as a result of physical modification of water permeability of the wall.
Like other antioxidant enzymes, catalase also plays a vital role in plant defense against salt-induced oxidative stress. To affirm this, Al-Taweel et al. (2007) examined the influence of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum 'Xanthi') and in transformed plants comprising the catalase gene katE from Escherichia coli. They observed that photoinhibition of PSII in leaf discs from both wild-type and katE transformed plants was increased due to salt stress, but the effect of salt stress was not so prominent in the transformed plants than in wild-type plants. The high tolerance of katE transgenic tobacco plants was suggested to be due to increased ability of the chloroplast's translational machinery of these plants to scavenge hydrogen peroxide under salt stress. Such a positive relationship between the activity of catalase and degree of salt tolerance has also been drawn in transgenic plants of Arabidopsis
(Willekens et al., 1997).
Of the different non-enzymatic antioxidant metabolites in plants, tocopherols are considered as potential detoxifying agents of ROS. Thus, to examine the role of tocopherols in stress tolerance of tobacco, Abbasi et al. (2007) have developed transgenic lines silenced for homogentisate phytyltransferase (HPT) and γ-tocopherol methyltransferase (γ-TMT) and examined the response of these transgenics to salt stress as well as to sorbitol stress andmethyl viologen treatments in comparison towild type. It was noted that the transgenics with low tocopherol content showed an increased sensitivity to all three stresses, indicating the positive role of tocopherols in stress tolerance, perhaps by detoxifying ROS.
Undoubtedly, engineering of genes coding for antioxidative enzymes has provided new insights into the role of these enzymes in plant cells in counteracting stress-induced ROS. In viewof a number of reports in the literature it is now evident that alteration in ROSscavenging systems may cause considerable modification in oxidative stress tolerance and hence changes in tolerance to abiotic stresses (Pastori and Foyer, 2002). Although in many studies the overproduction of SOD has been positively correlated with enhanced stress tolerance, some reports have shown an otherwise relationship. Such contrasting reports indicate that factors other than SOD overexpression may be involved in improved oxidative stress tolerance in transgenic plants. Thus, Kwon et al. (2002) suggested that a combined increase in SOD and APX levels may be necessary for enhanced
oxidative stress tolerance in some transgenic SOD plants. On the other hand, Miyagawa et al. (2000) have observed that transgenic tobacco plants expressing bacterial CAT showed enhanced SOD activity and inhibition of APX under oxidative stress. Similarly, while examining the role of an antioxidant enzyme, glutathione S-transferase, in protection of tobacco plants from salt-induced oxidative stress, Katsuhara et al. (2005) reported that the overexpression of the glutathione Stransferase gene though resulted in reducing the levels of ROS, the removal of ROS was not enough to influence the salt tolerance of tobacco plants. Furthermore, comparison of a wild-type and transgenic line of B. napus showed that the wild type B. napus accumulated high amounts of glutathione and cysteine upon exposure to salt stress, whereas B. napus transgenic plants did not show these antioxidative responses, although it had a high capacity to sequester sodium into the vacuoles, an important component of the mechanism of salt tolerance in most salt tolerant plant species (Ruiz and Blumwald, 2002).
Although manipulation of antioxidant genes seems to be a sound approach to counteract salt-induced oxidative stress, attempts to improve oxidative stress tolerance particularly by manipulation of a single antioxidant gene have not always been successful, presumably because of the need for a balanced interaction of protective enzymes and other metabolites (Zhang et al., 2000; Tseng et al., 2007). Thus, a balanced interaction of antioxidative enzymes as well as of other antioxidant metabolites may be vital to achieve a substantial improvement in plant stress tolerance. Furthermore, a question arises that up to what extent the gene manipulated contributes to overall stress tolerance at whole plant level as a multitude of phenomena related to the mechanism of salt tolerance are simultaneously operative in a cell or tissue. It is now explicit that the effects of salt stress on plants could be primary and secondary. Salt-induced oxidative stress is considered a secondary effect of salt, whereas osmotic stress and ionic effects are primary effects. Thus, while producing transgenic lines of a particular species for salt tolerance one must ensure the contribution of plant responses to the primary and secondary effects
Conclusion and future Perspective
Salt stress causes huge losses of agriculture productivity worldwide. Therefore, plant biologists aimed at overcoming severe environmental stresses needs to be quickly and fully implemented. Together with conventional plant physiology, genetics and biochemical approaches to studying plant responses to abiotic stresses have begun to bear fruit recently. Relevant information on biochemical indicators at the cellular level may serve as selection criteria for tolerance of salts in agricultural crops.
Major components of plant salt tolerance are osmoregulation and ion homeostasis, and tolerance to oxidative stress is considered as a secondary phenomenon. Thus, before proceeding to manipulation of genes for oxidative stress tolerance one should ensure the contribution of oxidative tolerance as well as other components of salt tolerance in a plant species of interest. Otherwise, the goals of achieving enhanced plant salt tolerance through engineering antioxidant genes will remain far and faulty. Since the salt tolerance trait is controlled by polygenes, it is naïve to expect its great complexity. The mechanism of salt tolerance even becomes more intricate in case when a crop tolerance varies at different phases of plant development. In this case, it is highly likely that the oxidative stress protection pathways predominant at one particular stage may not be so functional at other growth stages. Thus, understanding the extent of functioning of all components of salt tolerance including that of oxidative stress tolerance is necessary.
Plants growing on salt affected soils particularly in arid and semiarid regions are often simultaneously prone to multiple abiotic stresses such as high temperature, high light intensity, drought etc. as well as biotic stresses. Thus, it is imperative to elucidate how ROS metabolism is regulated during a combination of abiotic and biotic stresses. Knowledge of ROS regulation and antioxidant production is necessary for generating transgenics with altered levels of antioxidant enzymes and metabolites, because enhanced antioxidant production under one kind of stress may evoke tolerance to other stresses.