THE LEGUME-RHIZOBIA SYMBIOSIS UNDER SALT STRESS - A REVIEW

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1 Agric. Rev., 27 (1) : 1-21, 2006 THE LEGUME-RHIZOBIA SYMBIOSIS UNDER SALT STRESS - A REVIEW Ranju Singla and Neera Garg Department of Botany, Panjab University, Chandigarh , India ABSTRACT Salinity is one of the severe problems in worldwide agricultural production. Soil salinity limits the productions of both forage and grain legumes. Adverse effects of salinity are mediated through detrimental effects on the rhizobium - legume interactions that lead to the establishment of the nitrogen fixing symbiosis. Salt stress inhibits the initial steps of the rhizobia - legume symbiosis. For improvement of nodulation and symbiotic nitrogen fixation under saline environments, the answer lies in selecting and developing salt tolerant cultivars of legumes as well as rhizobium for effective symbiosis under salinity. This review focuses on the effect of salt stress on nodulation and symbiotic nitrogen fixation in legumes and to suggest future lines for alleviation of salinity effects on these processes. Soil salinity is a major agricultural problem, significantly reducing productivity of a broad range of crops (Lauchli and Epstein, 1990; Serrano and Gaxiola, 1994). Worldwide, about one third of the irrigated arable land is already salt affected and that portion is still expanding (Lazof and Bernstein, 1999). Each year approximately 10 x 10 6 ha of the all agricultural land is abandoned because of salinisation (Flowers and Yeo, 1995). In India, 7.04 million ha land is affected by salinity (Abrol and Bhumbla, 1971). The decline in the productivity of crops, in salt affected soils, is mainly caused by salt induced osmotic effects, ion toxicity and mineral perturbations in plants (Hu and Schmidhatter 1997; Volkmar et al., 1998; Lazof and Bernstein 1999; Lauchli, 1999; Hasegawa et al., 2000; Muhling and Lauchli, 2002). In fact, all major agronomic crops are relatively salt intolerant (Ashraf and O Leary, 1994; Lutts et al., 1995; Garg and Gupta, 1997; Garg and Gupta, 2001). High salinity causes both hyperionic and hyperosmotic stress effects, and the consequences of these can be plant demise (Yeo, 1998; Glenn et al., 1999; Hernandez et al., 2000, 2001; Rout and Shaw, 2001). Long time back, in the year 1953 Bernstein and Hayward wrote, An understanding of the physiology of salt tolerance of plants is important for an effective approach to the salinity problem, which is of increasingly widespread occurrence. Coupling and understanding of the genetic control of salt tolerance with this physiological approach adds further dimension of promising to lead to the development of salt tolerant crops. Tolerance to stress varies with the growth stage, climatic conditions as well as for different genotypes/cultivars of a crop within the same species. Different strategies have been adopted by soil scientists to solve the problem of salinity. The major strategy to overcome it includes reclamation of salt affected soils by using preventive and curative measures; but the cost in terms of money and energy is high. A possible alternative, the biotic approach, is to lay emphasis on the selection and development of varieties which give minimum depression in yield when grown under saline conditions. Selection of high salt tolerant genotypes within a species, in comparison to relatively salt sensitive ones, highlights the prospects for improvement through conventional selection and breeding technique. A prerequisite in selection and breeding for salt tolerance is the presence of genetic variations for tolerance in gene pool of the species. Interspecific, intra-

2 2 AGRICULTURAL REVIEWS specific and intra-cultivar variations for a character provide scope for their improvement (Johnsen et al., 1990; Ashraf, 1994). Legumes belonging to family Fabaceae, the third largest family of angiosperms, with three subfamilies comprising 650 genera and more than 18,000 species (Polhill, 1994), rank next to cereals in importance as food for human beings and animals. In addition to their nutritive values, legumes have considerable importance in agriculture for their ability to improve soil fertility by fixing large amounts of atmospheric nitrogen. Legumes have long been recognized to be very sensitive to salinity (Lauchli, 1984). Amongst grain legumes lentil, chickpea, mungbean, pigeonpea etc, widely grown in the subcontinent because of their importance as pulse crops, are considered to be relatively salt sensitive crops as compared to soybean. Despite their great importance, very little work has been done on the improvement of salt tolerance in legume crops. Differences in salt tolerance, at the cultivar and genotypic levels, have been reported in several leguminous crops (Lauchlii, 1984; Dua et al.,1998; Subbarao and Johansen, 1994; Serraj et al., 1998, 2001) and exploitation of genetic variability in cultivated species offers the possibility of developing salt tolerant crops (Epstein et al., 1980). However this variation has not been evaluated thoroughly and systematically for most of the leguminous crops. Physiological responses of plants to salinity are one of the most studied subjects in plant physiology (Flowers et al., 1977; Wyn Jones, 1981; Greenway and Munns, 1980; Flowers, 1985; Munns and Termaat, 1986; Epstein and Rains, 1987; Cheeseman, 1988; Munns, 1993; Hasegawa et al., 2000). Progress towards achieving salinity tolerance in crops can be made only when physiological and biochemical processes for tolerance are understood (Shannon, 1985; Yeo and Flowers, 1989; Shannon and Noble, 1990; Cuartero et al., 1992; Munns, 1993) Inter-specific, intraspecific and intra-cultivar variation is of prime importance for the improvement of salt tolerance through selection and breeding. The existence of genetic variability in the sensitivity of N 2 fixation and to salt among legume species and cultivars may be useful to further elucidate the NaCl inhibition symbiotic nitrogen fixation to select optimal Rhizobium-legume symbiosis for agricultural production in soils subjected to salinity (Serraj et al., 2001; Serraj, 2002). During the last two decades, a number of workers like Lauter and Munns, 1982; Olmos and Hellin, 1996; Adb El Samad et al., 1997; Dua and Sharma, 1997; Dua, 1998; Soussi et al., 1999; Sleime et al., 1999 have tried to identify the traits attributing towards salinity resistance amongst the different legume species. However, no consistent and specific correlations were identifiable. In order to have a better understanding of the influence of salinity stress and the mechanism of salinity resistance, it is needed to study the morphophysiological and biochemical behaviour of different cultivars/genotypes of a species and to visualize the traits associated with salinity tolerance or susceptibility with the help of experimental evidence. In the light of most recent literature available, the effect of salt stress on various metabolic activities of legumes has been reviewed under the following heads: 1. Effects of salt stress on germination, growth and metabolic biology. 2. Effects of salinity on nitrogen metabolism. 3. Ameliorative measures to salt stress. 1. Effects of salt stress on the germination, growth and metabolic biology of legumes Salt accumulation in the soil of arid regions and irrigated fields often described as salinity, impairs plant growth. Salinity stress influences plants at every stage of the lifecycle and stimulates water stress in many ways. The main inhibitory effect of salt stress is osmotic.

3 Sodium chloride is usually present in the soil and, although rather innocuous in its inorganic constituents, influences many membrane functions and induces membrane ultra structural changes. Growth of plants in the presence of sodium chloride may impair cellular ion homeostasis, characterized by high K+ and low Na+ cytoplasmic content. Increased salinity can affect plant growth by : the imposition of water stress through increased osmotic potential of the rooting material (Levitt,1980; Yeo,1983; Hale and Orcutt, 1987) or the accumulation of ions or other possible toxins in the soil or in the plant tissue (Flowers,1985; Munns, 1988, 1993; Jacoby, 1994). 1.1 Cellular water relations: Legumes have long been recognized as either sensitive or only moderately resistant to salinity (Delgado et al., 1994). At cellular level many of the physiological effects of salinity on shoot and leaf growth are typical of a water stress which necessitates a corresponding adjustment in the osmotic potential of the plant cells to sustain water uptake. According to Neumann et al. (1988) salt stress initially inhibits leaf expansion through reduced turgor, and may infact eventually result in increased cell wall extensibility, which counteracts the negative effects of low turgor. In the presence of salt, cell wall extensibility of the growing region may decrease (Cramer, 1992; Nonomi et al., 1995). Wu et al. (1997) suggested that ABA modulated the expression of enzymes affecting plasticity of the cell wall. These results suggest that the hormonal balance is able to control the cell wall expansion. Crop plants show some degree of tissue osmotic adjustment in response to either salinity or drought (Mastuda and Riazi, 1981). Accumulation of compatible solutes in response to stress is a metabolic adaptation found in a number of stress tolerant, often unrelated taxa, suggesting convergent evolution for this trait (Yancey et al., 1982). Vol. 27, No. 1, The osmoprotectants are synthesized in response to stress and are localized in the cytoplasm (McCue and Hanson, 1990; Delauney and Verma, 1993; Louis and Galinski, 1997; Glenn et al., 1999) and lead to turgor maintenance for the cell under osmotic stress. A variety of osmoprotective compounds such as proline, glycine betaine, various sugars (sucrose and fructose), sugar alcohols (glycerol, methylated ionsitols) and complex sugars (trehalose, raffinose, fructose) accumulate in response to osmotic stress and act as compatible solutes i.e. they do not inhibit normal metabolic reactions (Pharr et al., 1995; Piqueras et al., 1996; Hasegawa et al., 2000). 1.2 Ionic Relations: The accumulation of toxic salts in tissues has frequently been postulated as another limiting factor for growth under salt stress. Although many biochemical functions require specific inorganic ions, increasing the concentrations of these ions above normal intracellular concentrations may lead to disruption of metabolic functions by reducing the activities of enzymes (Yancey et al., 1982). Leopold and Willing (1984) suggested that NaCl could cause leakage of solutes from soybean leaf tissue which could interact with cellular membranes. According to Greenways and Munns (1980) older leaves tend to have higher levels of Na + and Cl - than younger leaves because of continuous accumulation of these ions over time due to transpiration. Salt accumulation in the plants leads to reduced longevity of mature leaf tissue. Dale (1986) explained reduced leaf size due to salt stress on the basis of limited assimilate supply. Munns (1993) argue that salt concentrations in older leaves accelerated their death leading to reduced assimilate or hormone export to the rest of the plant. Nonami et al. (1995) suggested direct effect of Na + on cell expansion. Volkmar et al. (1998) explained reduced production in leaves

4 4 AGRICULTURAL REVIEWS on the basis of reduced storage capacity in the shoots in the form of cell vacuoles to accommodate salts arriving from the roots, leading to further salt toxicity. Lauchli (1999), Santa- Maria and Epstein (2001) reported the importance of maintenance of adequate net uptake of K + by plants at high Na + and this sustained Na + /K + selectivity as a physiological marker for the ionic component of salt stress. Hasegawa et al. (2000) suggested that signaling cascades presumably function in intercellular coordination or regulation of effectors genes in a cell-/tissue-specific context required for tolerance of plants. Zhu (2002) has proposed that salt stress signal transduction consists of ionic and osmotic homeostasis signaling pathways detoxification (i.e. damage control and repair) response pathways, and pathways for growth regulation. He further states that osmotic stress activates several protein kinases including mitogen-activated kinases which may mediate osmotic homeostasis and/or detoxification responses. A number of phospholipid systems are activated by osmotic stress, generating a diverse array of messenger molecules, some of which may function upstream of the osmotic stress activated protein kinases. Munns (1993) has proposed another biphasic model of growth response to salinity. In the first phase toxic ions increase especially in the mature leaves. These fully expanded leaves often show necrotic leaf tissue, while in younger expanding leaves salt induced water deficit leads to Ca 2+ deficiency symptoms (Fortmeier and Schubert, 1995). Therefore Na + accumulation in leaves, particularly in the leaf apoplast, could be responsible for Na + toxicity in leaves (Volkmar et al., 1998). Oertli (1968) originally proposed that salt lead to the death of leaves by dehydration of leaf cells and turgor loss. Na + accumulation in the leaf apolast may also occur because of stimulation of the plasma membrane ATPase under salt stress (Niu et al., 1995), which may lead to an increased Na + efflux from the cytoplasm into the apoplast or by incoming salt from the root which could no longer be sequestered into the cell vacuoles (Munns, 1993).Muhling and Lauchli (2002) also supported the hypothesis that salt accumulation in the leaf apoplast could be responsible for the death of leaves in plants exposed to salinity. 1.3 Mineral Composition and Ion Uptake: The simultaneous presence of salts and nutrients elements in the root can influence nutrient uptake by plants and thereby affect their chemical composition. Murumkar and Chavan (1986) found increased concentrations of Na, Cl, P in pods and seeds of chickpea with a decrease in the concentrations of Ca in pods and Fe, Mn, Mg in seeds under increasing salt. Increasing levels of salinity significantly reduced the uptake of P, Zn, Fe in chickpea (Dravid and Goswami, 1987). Concentrations of Ca, Mg, Na increased and that of K and B decreased with increasing salinity ( dsm -1 ) (Yadav et al., 1989). Elshiekh and Wood (1989) while working on specific ion effects in chickpea found chloride ions of Na, K, Mg to be more toxic than the corresponding sulphate ions. According to Dhingra et al. (1994) salinity caused accumulation of Na + and Cl - in the seeds of chickpea but did not affect K + content. Cordovilla et al. (1995) reported higher salt sensitivity of faba bean and pea, which accumulated more Mg 2+, Ca 2+ in shoots and Na +, K + in roots as compared to common bean and soybean showing the direct relationship between accumulation of Na + in shoots and plant salt sensitivity. Mamo et al. (1996) found chloride concentration (mg/g) in chickpea plant parts at salt levels (0-8 dsm -1 ) 2-5 times that of Na with significant reduction in K + concentration. Zayed and Zeid ( ) reported reduced mineral uptake in mungbean under salt stress rather than PEG induced water stress due to maintenance of higher succulence under salt stress. Khan et al. ( )

5 Vol. 27, No. 1, studied the physiological responses of alfalfa (Medicago sativa L.) to salinity (100 mm NaCl) and some inorganic nutrients (K +,Ca 2+, N as NO 3- ) and reported that inclusion of these nutrients in plant nutrient medium in combination or alone brought the marked stimulation in control plants and moderated the salinity caused reductions in NaCl treated plants. On the basis of their study on four chickpea genotypes belonging to the tolerant and susceptible groups (salt stress). Singh and Singh (1999a) reported lower Na and higher K in the shoots of tolerant genotypes as compared to the susceptible genotypes. Sekeroglu et al. (1999) also found that salinity caused decreased K content while increased Na content of chickpea seedlings. Baalbaki et al. ( 2000) while working on ionic relations in chickpea cultivars found that salinity affected shoot Na + and Cl - contents but nodulating plants had higher shoot to root ratio and higher levels of Na + and K + than plants supplied with mineral nitrogen. Na concentration increased significantly in all plant parts in alfalfa (Medicago sativa L.) as the level of salinity treatments increased ( dsm -1 ) while P, K, Ca, Mg concentration decreased; the roots accumulated significantly more Na than other plant parts (Esechie et al., 2002). 1.4 Germination: Soil salinity can significantly inhibit seed germination and seedling growth, not only in glycophytes, but also of halophytes, due to combined effects of high osmotic potential and specific ion toxicity. The reduction in germination under saline conditions could be attributed to the increased osmotic pressure of soil solution which reduces the water absorption rate, leading to moisture stress in the seeds and reduced mobilization of food reserves. This inhibition of reserves mobilization could be because of the effects of salts on the enzyme responsible for hydrolysis and effects on the translocation of reserve hydrolysis products from the storage organs to the embryo axis (Garg and Gupta, 1998). Mehta and Bharti (1983) observed that chloride and sulphate salinity (4 to 12 dsm -1 ) affected seed germination in gram and decreased the fresh and dry weight of the embryo axis. Sheoran and Garg (1983) found that Na 2 SO 4 significantly decreased germination in gram while NaCl, KCl, K 2 SO 4 only delayed it. Siddiqui and Krishnamoorthy (1986) observed decreased length and fresh and dry weights of the embryo axis of cowpea and chickpea with increasing salinity. Saxena et al. (1989) found relatively higher degree of salt tolerance with a mixture of salts in terms of germination in alfalfa and the relative order of salt tolerance was; NaCl > CaCl 2 > Na 2 SO 4 > Na 2 CO 3 > NaHCO 3. Dua (1992) while working on 20 genotypes of chickpea observed that sensitivity of all genotypes increased with plant growth and higher salinity levels. Further, chlorides were found to be more toxic than sulphates. Mamo et al. (1996), while working on chickpea and lentil observed differences amongst different varieties in response to NaCl (0-8 dsm -1 ) application, with lentil showing relatively higher percentage emergence as compared to chickpea. Zurayk et al. (1998) also observed significant reduction in the germination of 18 cultivars of chickpea, calculated as speed of germination index (SGI) as well as early seedling growth but the response varied with the type of cultivars as well as the salinity levels. Sekeroglu et al. (1999) and Khalid et al. (2001) observed decreased germination, freshweight, radicle and plumule length and K content with a concomitant increase in the Na content under salinity in chickpea. Similar effects were reported by Dash and Panda (2001) in black gram (Phaseolus vulgaris L.). The relative effects of salinity on germination and early seedling growth in four legume species namely pigeonpea, chickpea, mungbean and soybean varied under saline conditions with chickpea

6 6 AGRICULTURAL REVIEWS proving to be most salt susceptible and soybean most salt tolerant (Garg and Dua, 2000). Promila and Kumar (2000) observed reduced seed germination in mung bean under salinity. With an increase in salinity (NaCl) concentrations, there was corresponding decrease in imbibitions and germination of bean cultivars and the variability amongst these cultivars gave scope for selection of cultivars tolerant to salinity (Moreno et al., 2000). Similar varietal variability s in salinity tolerance of seven alfalfa varieties and two chickpea cultivars at the germination stage have been reported by Maiti et al. (2002) and Esechie et al. (2002), respectively. Soltani et al. (2002) studied the interactive effects of seed size and salinity on germination and seedling growth in chickpea and found that larger seed did not have any advantage in producing more vigorous seedlings under saline condition. Demir and Kocacalinian (2002) reported decreased seedling growth under NaCl treatment and application of proline helped alleviate salinity stress in bean (Phaseolus vulgaris L.) seedlings. The salt tolerance at germination and early seedling growth is important because the initial stand determines the ultimate production to a large extent. 1.5 Growth And Development: Salinity is a major constraint in agriculture and adversely affects germination of seeds, plant growth and metabolism (Dua, 1992; Zurayk et al., 1998). In legumes, salt stress imposes a significant limitation on productivity. Not only the varying concentration of salts but the different types of salinity have different aspects on plant growth. Manchanda and Sharma (1989) reported that chickpea growth was reduced by sodium salts at very low concentration but sensitivity was high when the saline soil has a high concentration of sulphate and/or low concentration of chloride. Hafeez et al. (1988) found that increasing NaCl salinity decreased the dry matter yield of Vigna radiata L. irrespective of the stages of plant growth. Sharma et al. (1990a, b) investigated the effect of chloride and sulphate salinity in chickpea and found chloride salinity to be more deleterious than sulphate salinity. Wignrajah (1990) found that low salinity stress (48 mm NaCl) resulted in delayed development of leaves as well as plant growth with higher negative effects on shoot growth than root growth in Phaseolus vulgaris L. Increased leaf area ratio (LAR) and net assimilate rate (NAR) were identified as major physiological traits for salinity tolerance. Similar negative effects on root and shoot growth has been reported in chickpea by Elsheikh and Wood (1990) and in maize and soybean by Shalhevet et al. (1995). Delgado et al. (1994) and Cordovilla et al. (1995) compared the effect of salinity on growth and productivity and found pea, fababean to be significantly affected and soybean and common bean to be moderately effected. Zaidi and Singh (1995) reported that salinity inhibited leaf growths as well as net assimilate rate and relative growth rate of the soybean plants. Dua and Sharma (1995) studied the relative salt tolerance of desi and kabuli genotypes of chickpea and found kabuli genotypes to be more salt tolerant than desi. Mamo et al. (1996) observed significant differences among different varieties of chickpea and lentil in their response to NaCl application with few varieties showing some degree of superiority in terms of mean relative shoot and root dry weights, and grain yield over the others. Salinity (100 mm NaCl) caused substantial reduction in leaf area, relative growth rate in alfalfa (Khan et al., ). Rogers et al. (1998) found lucerene to be moderately tolerant to Na 2 SO 4 predominated salinity that to NaCl dominated salinity and the dry matter production was negatively correlated with shoot concentration of specific ions like Na +, C - and S 2-. Zurayk et al. (1998) reported significant reduction in the dry weight of above ground biomass in chickpea on

7 Vol. 27, No. 1, treatment with NaCl +, Na 2 SO 4 salts (0.5 dsm -1, 3dSm -1, 6dSm -1, 9dSm -1 ). Similarly, Soussi et al. (1998, 1999) reported decline in plant growth of Cicer arietinum L. by 100 mm salt concentration. Cordovilla et al. (1996, 1999) also reported the decline in shoot and root weight on treatment with salt and salinity affected root growth more than shoots in faba bean, thus increasing the root to shoot biomass. Growth inhibition by salt in common bean plants proved significant in the experiments carried out by Ferri et al. (2000). The effect of salt was more pronounced in the second sampling, taken during the reproductive period, which could be explained by the excessive salt accumulation. Al-Khanjari et al. (2002) observed variation in dry matter yields of alfalfa (Medicago sativa L.) cultivars to salt tolerance. Salinity significantly reduced both shoot dry matter and root volume. 1.6 Metabolic alterations: A number of investigators have studied metabolic changes induced by salt stress in legumes with a view to understand the physiological and biochemical basis of salt tolerance. 1.6 a. Amino acid, Protein and Carbohydrate Metabolism: Salinity is known for its depressive effects on metabolic pathways and energy generating normal growth processes. Mehta and Bharti (1983) found that both chloride and sulphate salinity increased the soluble carbohydrates in germinating seeds of chickpea but free aminoacids were reduced. A NaCl tolerant callus line of chickpea accumulated free proline in response to increasing NaCl concentrations and its contents markedly suppressed upon substituting KCl for NaCl (Pandey and Ganapathy, 1985). Murmukar and Chavan (1986) reported that increasing NaCl salinity decreased the protein and starch content of the seeds while the sugar and free proline contents increased the pod shell in chickpea followed by a reduction in photosynthetic carbon assimilation. In chickpea cultivars, both chloride and sulphate salinity reduced carbohydrates, starch and protein content of leaves, however, aminoacids and proline increased with increasing conductivity (Sharma et al., 1990a,b). Dhingra and Sharma (1993) on the basis of their analysis on two distinct types of seeds of mungbean healthy and shriveled found that total soluble sugars (including reducing sugars), proteins, free amino acids and proline were much lower in shriveled seeds than the healthy ones under saline conditions. Dhingra et al. (1994) observed that seeds of three promising genotypes of chickpea (ICCV 88102, H82-2 and C-235) differed in accumulation of soluble sugars and starch. With salinity, starch and protein content decreased in the chickpea seeds with no effect in the sugar content (Dhingra et al., 1995). Singh and Singh (1995) reported a continuous decline in reducing, nonreducing, total saccharides, total protein contents with increasing sodicity, though the free proline content was enhanced in all three genotypes of pea. Durgaparsad et al. (1996) reported that soybean seedlings germinated under different NaCl salinity increased protease activity, aminoacids and proline accumulation though it decreased the utilization of reserve protein of the cotyledons. In chickpea cv. ICCV 88102, lower salinity at sowing increased protein content marginally whereas higher salinity decreased it substantially (Dhingra et al., 1996) Zaidi and Singh (1995) reported proline accumulation in soybean plants under salinity. The chloride salinity declined starch content in the chickpea calli (Sangwan et al., 1997). Garg et al. (1998) reported that salinity induced changes in levels of certain leaf metabolites (starch, reducing sugars, total chlorophyll, soluble protein, free amino acids and free proline) in clusterbean and increasing salinity led to significantly higher metabolic derangements which were particularly more pronounced at the flowering stage, as compared with other stages of

8 8 AGRICULTURAL REVIEWS growth. Salt reportedly boosted proline, amino acid and carbohydrates in the leaves of chickpea. (Soussi et al., 1998). Singh and Singh (1999a) found that tolerant genotypes of chickpea had higher proline content in shoots under salinity as compared to the susceptible genotypes. Paneerselvam et al. (1998) reported that NaCl stress caused accumulation of proline and aminoacids and decreased protein and nucleic acid content in soybean seedlings, but addition of triadimefon restored the growth and increased the protein, amino acid and nucleic acid content. Bean (Vicia faba) plants when subjected to salinity had decreased soluble and hydrolysable sugars, soluble proteins and enhanced total free amino acids (Gadallah, 1999). Promila and Kumar (2000) reported that amylase activity in the cotyledons of Vigna radiata was progressively reduced with increasing NaCl concentration but the increased soluble sugar content in the cotyledons indicated that sugars were not limiting for mungbean seeding growth under salinity. Mansour (2000) observed accumulation of nitrogen containing compounds like amino acids, amides, proteins, quaternary ammonium compounds (QAC) and polyamines in legume plants exposed to salinity stress. Dash and Panda (2001) and Rai (2002) reported increased proline content in legume plants on subjection to salinity stress. Demir and Kocacalikan (2002) reported that NaCl decreased seeding growth in bean seedlings cultured invitro, while proline added to control seedling did not change seedling growth but decreased chlorophyll and increased protein content. Soybean showed higher tolerance towards saline condition as compared to gram and index for salt tolerance seemed to be directly related with higher levels of sugars, amino acids, proteins and nucleic acid (Garg, 2002). 1.6b. Carbon Metabolism and Carbon Catabolism: The effect of salt stress on carbon metabolism is related to the salt concentration in the photosynthetic tissues and response of enzymes like ribulose 1-5 bisphosphate carboxylase oxygenase and phosphoenolpyruate carboxylase. Salinity may affect the overall process of photosynthesis at different points, either inhibiting enzyme activity or by altering it. The effects of salinity on photosynthesis can be both stomatal (Brugnoli and Lauteri, 1991) and non-stomatal (Seeman and Chritchley, 1985). Salinity may result in decreased quantum efficiency of carbon dioxide uptake (Seeman and Chritchley, 1985), change in the ionic relations of the chloroplast (Long and Baker, 1986) or change in photochemical reactions (Reddy et al., 1992). Seeman and Critichley (1985) reported inhibition of Rubisco activity by the salt stress in Phaseolus vulgaris, which may be due to sensitivity of this enzyme to chloride ions. Bekki et al. (1987) reported diminshed respiratory capacity of the Medicago nodules under sodium chloride stress. Plaut (1990) also reported decline in both photosynthesis and Rubisco activity when salt concentration exceeded 65 mm in bean. Wignarajah (1990) found that low salinity stress resulted in delayed development of the leaves and, further, increased leaf area ratio (LAR) and net assimilate rate (NAR) were identified as major physiological traits for salinity tolerance. Brugnoli and Lauteri (1990) observed reduction in leaf area development and stomotal conductance under salinity. NaCl reduced photosynthesis by 170 mol m -3 in the salt tolerant cultivar (Kapulink and Hever, 1991). Irigoyen et al. (1992) reported inhibition of electron transport and photophosphorylations under saline conditions in alfalfa. Malate concentration reportedly diminished with salt stress, but under these conditions nodule cytosolic phosphoenolpyruvate carboxylase

9 activity and bacteroid malate dehydrogenase increased in the nodules of pea (Delgado et al., 1993). Khan et al. (1994) studied the interaction between salinity and nitrogen forms in alfalfa (Medicago sativa) and observed that photosynthesis was more significantly reduced in ammonium than in nitrate fed plants. Further, the promotive effect of nitrogen on photosynthesis in saline as well as in non-saline conditions could be attributed to enhanced synthesis and availability of carbon assimilatory enzymes and cofactors required for optimal photosynthesis. Awada et al. (1995) also reported reduced photosynthetic rate by all levels and types of sodium salts in Phaseolus vulgaris L. Zaidi and Singh (1995) reported reduction in total chlorophyll, chlorophyll a/b ratios, as well as NAR under salinity in soybean plants. Fernandez-Pascual et al. (1996) observed increased oxygen diffusion resistance with 100 mol m -3 NaCl in white lupin. Five day old Vigna radiata seedlings exposed to 200mM NaCl significantly increased the level of NADH, H + with marked decrease in the e - transport activities than control (Sarandhi et al., 1996). Sudhakar et al. (1997) observed salinity shock caused decline in the activities of Ribulose bisphosphate carboxylase and further, NaCl was more toxic to the enzyme activity as compared to Na 2 SO 4. Reduced photosynthesis rate was reported in two chickpea genotypes under increasing salinity level (Sharma, 1997). Zayed and Zeid ( ) and Garg et al. (1998) observed significant reduction in chlorophyll content in Vigna radiata and Cyamaposis under salt stress, respectively. Singh and Singh (1999b) reported an increase in carotenoids in tolerant genotypes of chickpea whereas a decrease was observed in the susceptible ones under salt stress when analysed at the seedling stage. Soussi et al. (1998) while working in the Dept. of Biologia Vegetal, Univ. of Granada, Spain reported that photosynthetic carbon assimilation process, leaf chlorophyll content Vol. 27, No. 1, in chickpea was greatly depressed by the high levels of salt. Moreover, the activity of leaf ribulose, 1, 5 biphosphate carboxylase declined with an increase in the nodule phosphoenolpyruvate carboxylase and malate dehydrogenase activity under salinity at the first harvest, and a decrease at the later stage. Gadalladh (1999) observed reduced chlorophyll content in Vicia faba under salinity which increased upon application of proline and glycinebetaine. Soussi et al. (1999) reported that photosynthesis was more affected by salt in Pedrosillano (sensitive) cultivar of chickpea than in ILC 1919 (tolerant) and phosphoenolpyruvate carboxylase(pepc), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH) activity in the nodules cytosol was higher in the tolerant cultivar under saline conditions which could improve regulation of oxygen diffusion. A significant increase in net photosynthetic rate (P N ) at low salinity and reduction at higher salinities was observed in alfalfa genotypes (Anand et al., 2000) which was primarily due to reduction of stomatal conductance. Ferri et al. (2000) observed decline in PEPC, MDH, ADH, ICDH (isocitrate dehydrogenase) activities in the nodule cytosol by the salt treatments, while in bacteroid cytosol, the enzyme activities increased at high salt concentrations. Moreover the respiratory capacity of bacteroids was depressed by salt which diminished further with plant age. Al-Khanjari et al. (2002) studied the variabilities amongst alfalfa cultivars on the basis of chlorophyll content to salinity and reported that magnitude of decreases in chlorophyll concentration in cultivars was in line with their tolerance to salinity. Reduced chlorophyll content under NaCl was also reported in Phaseolus vulgaris by Demir and Kocacalikan (2002). Respiration and nitrogen fixation in legume root nodules is limited by the rate at which oxygen (O 2 ) from atmosphere enter

10 10 AGRICULTURAL REVIEWS nodules. A number of workers have reported lowered nitrogenase activity of legume root nodules due to limitation of O 2 supply to bacteroids (Witty et al., 1984; Minchin et al., 1986; Caroll et al., 1987; Atkins et al., 1988). Dakora and Atkins (1990), reported decrease in nitrogen fixation at PO 2 below 5% in Vigna unguiculata nodules which may be due to lower nodulation and nodule mass and at po 2 above 60%, to a fall in specific nitrogen fixing activity of nodules. For nitrogenase activity, regulation of O 2 supply to infected cells was necessary and there existed a variable diffusion barrier within the inner cortex (Hunt and Layzell, 1993). Iannetta et al. (1995) studied time course of changes involved in the operation of the O 2 diffusion barrier in Lupinus albus L. (white lupin) nodules and found increased glycoproteins within the cell walls following exposure to 50% O 2 for 30 min. Suganuma and LaRue (1995) reported decrease in acetylene reduction activity in soybean plants on exposure of roots to 100% O 2 and, further, there was accumulation of succinate, malate, alanine in nodules on treatment with O 2. Bergersen (1997) observed from studies on soybean bacteroids that nitrogenase is converted to less active but robust form, in the presence of O 2 (in excess of about 70 nm), protecting from inactivation by excess O 2. He further observed that, respiration by large numbers of host mitochondria in the periphery of infected nodule cells, adjacent to gas - filled intercellular spaces, plays an important role in maintaining a steep gradient of O 2 concentration in this zone. Rao (1999) studied the effect of elevated CO 2 levels and temperature on Arachis hypogaea L. and reported stimulated plant growth and biomass production under elevated CO 2 ( ppm) + elevated temperature (40 o C) than ambient CO 2 ( ppm) + control temperature (35 0 C). Lundquist (2000) on the basis of interactive effect of oxygen and short term nitrogen deprivation in Alnus incana root nodules, reported decline in nitrogenase activity. Shrivastva et al. (2001) repeated increased nodulation, nitrogense activity and root growth under elevated carbon dioxide (600+50ppm) in Phaseolus radiatus. 1.6c. Enzyme Activities: Plant growth is the result of many integrated and regulated physiological processes. Salinity, at the whole plant level, effects changes in photosynthesis and in carbon, and nitrogen metabolism and increase and decrease in various enzyme activities which alter the growth and development of the plant (Alam, 1990; Yang et al., 1990). Khatri et al. (1985) reported decrease in starch degradation and its utilization in the embryo axis of Cicer arietinum L. under salt stress which was closely correlated with decrease in soluble sugars and α -amylase activity in chickpea genotypes differing in salt tolerance. Dhingra (1994) reported higher succinate dehydrogenase activity which implied that major fraction of soluble sugars produced due to higher hydrolytic activities in response to salinity was utilized as a substrate for respiration and this extra metabolic energy was likely to be consumed in the process of osmotic adjustment and survival under adverse conditions. Filho Eneas et al. (1995) observed negative effect on the activities of α and β glatcosidases under 100 mm NaCl treatment in cotyledons of Vigna unguiculata. Olmos and Hellin (1996) reported higher presence of enzymes related to sugar metabolism (glucokinase, fructokinase and acid invertase) in pea response to NaCl salinity. Lower activity of polyphenol oxidase (PPO) enzyme in the embryos and food tissue of bean under salinity was reported by Kabar et al. (1997). Sangwan et al. (1997) observed continuous declension in alpha-amylase in two genotypes of gram on subjection to salt. Zayed and Zeid ( ) reported decline in the activity of α-

11 amylase and protease in germinating Vigna radiata seeds during 3-d salt stress and increase in the activity of hydrolytic enzymes during 10 day stress. Guerrier et al. ( ) studies the relationship between proline contents and activities of proline biosynthesis [ornithine transaminase; NAD(P)H-pyroline carboxylase reductase], of proline catabolism [NAD(P) proline dehydrogenase] and of NAD kinase, and reported that these enzymes were clearly NaCl-regulated which resulted in proline accumulation in soybean calli. Arachis hypogaea L. were able to grow at high concentration of NaCl due to alteration in gene expression and induction of nine different esterase isoenzymes in the embryos of seeds germinated in 105 mm NaCl (Hassanein, 1998). Soussi et al. (1999) reported inhibitions of enzymes of sucrose breakdown by NaCl in chickpea cv. Pedrosillano but in cv. ILC 1919 a rise in alkaline invertase was observed, which could compensate for the lack of the sucrose synthase hydrolytic activity. Malate dehydrogenase and alcohol dehydrogenase activity was inhibited by salinity in the nodule cytosol of Phaseolus vulgaris L. (Ferri et al., 2000). Promila and Kumar (2000) reported reduced amylase activity in Vigna radiata seeds with increasing NaCl concentration. Dash and Panda (2001) observed that with the increase in NaCl concentration and duration of stress, catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities decreased, while proline increased. Singh et al. (2001) studied variability in the four chickpea genotypes under salinity and found higher activities of amylase, protease, peroxidase and catalase in the tolerant genotypes. Similar variabilities were reported in lentil (Lens culinaris) genotypes by Singh et al. (2001). Panda (2001) reported increase in the activity of antioxidative enzymes like catalase, guaiacol peroxidase and superoxide dimutase in root and shoot tissues of Vigna radiata L. under salinity stress. Malencic et al. (2003) also Vol. 27, No. 1, studied stress tolerance parameters in the different genotypes of soybean and reported high superoxide dimutase activity and low lipid peroxidation in the tolerant genotypes. Kaur et al. (2003) reported higher amylase activity, increased activities of sucrose synthase (SS) and sucrose phosphate synthase (S PS) in the cotyledons and shoots of Kinetin treated salt stressed chickpea seedlings which could be responsible for an increase in sucrose turnover in the shoots of stressed seedlings. 2. Effects of salinity on Nitrogen metabolism Legumes have the innate ability to form symbiotic association with soil bacteria and rhizobia and to fix atmospheric nitrogen. But symbiotic nitrogen fixation is particularly sensitive to environmental stresses like salinity whose effects are mediated through detrimental effects on nodulation and symbiotic nitrogen fixation. Salinity may differentially affect various phase of legume - Rhizobuim symbiosis namely: Rhizobium survival and infection of the host, nodule initiation and development, nodule functioning or nitrogen fixation process as well as growth of the host legume and its ability to maintain a constant supply of photosynthates and nutrients to the root nodule. 2.1 Nodulation and Nitrogen Fixation: The adverse effect of salinity on legume - Rhizobium symbiosis include decreased nodulation, nodule mass and decreased nitrogen fixation. Campbell et al. (1986) observed substantial reduction in nitrogen fixation as well as total nitrogen content in snap bean plants grown under saline conditions. Hafeez et al. (1988) also observed that salinity reduced nodule formation in Vigna radiata, but when the nodules were formed salinity did not affect their functioning provided the plants maintained reasonable photosynthetic activity. Salinity induced depressive effects on

12 12 AGRICULTURAL REVIEWS nodulation, leghemoglobin (Lb) content, nitrogenase and peroxidase activities which were metabolically regulated and operated through the intervention of some key regulatory substances in mungbean (Garg et al., 1988). Pessarakli and Zhou (1990) studied variabilities in three cultivars (Tender Improved, Slim green, Dentucky Wonder) of green bean (Phaseolus vulgaris) for nitrogen fixation under salt stress and found that salt stress significantly decreased total N content, per cent of N fixed and amount of total N fixed by plants in slim green. Sharma et al. (1990 b) reported that both chloride and sulphate salinities reduced nodule dry weight, nitrogenase activity, nitrogen percentage in two cultivars of chickpea and magnitude of declension enhanced under Cl - salinity as well as with the rise in the salinity level. Elsheikh and Wood (1990) reported that in chickpea cultivar inoculated with salt tolerant Rhizobium strain Ch 191, salinity decreased total nodule number/plant nodule weight and average nodule weight. Further Rhizobium was able to form an infective and effective symbiosis under saline and non-saline conditions suggesting more salt sensitivity of host chickpea as compared to the Rhizobium. Subbarao et al. (1990), Subbarao and Johansen (1994) observed significant differences among Cajanus cajan L. and different Rhizobium strains in their ability to nodulate and fix nitrogen under saline conditions, and observed that the early stages of establishment of symbiotic system was sensitive to salinity which became efficient in fixing nitrogen after the establishment of symbiosis. In alfalfa, on subjection to NaCl stress, total organic acid concentration in nodules was depressed, while it induced a large increase in the amino acids and carbohydrate pools (Fougere et al., 1991). It was further observed that within the amino acids; proline and amongst carbohydrates; pinitol concentration increased significantly thus suggesting their contribution to salt stress. Qifu and Murray (1993) observed negative effects of SO 2 concentrations on nodule number/dry weights, nitrogenase activity and reduced shoot and root nitrogen contents which could be ameliorated by NaCl salinity probably due to decreasing SO 2 uptake through stomatal closure. Reduced nodule weight and N 2 -ase activity was reported in snapbeans (Phaseolus vulgaris ) under NaCl salinity (Akhavan - Kharazian et al., 1991) However, addition of calcium to NaCl treatment increased these parameters and positive effect of calcium on nitrogen fixation was attributed to maintenance of selective permeability of membranes. Delgado et al. (1993) observed reduced ARA and leghemoglobin (Lb) content in the nodules of pea along with decreased malate concentration in bacteroids and cytosol of pea under NaCl stress with an increase in total soluble sugars due to inhibition in the utilization of the carbohydrates within the nodules. This decrease in leghemoglobin (Lb), respiratory capacity of bacteroids and malate concentration in nodules, induced by salt stress, were identified as some of the important mechanisms involved in inhibitory effect of salinity on nitrogen fixation. Delgado et al. (1994) reported depressive effect of saline stress on dry weight and ARA of nodules as well as the respiratory capacity of bacteroids in pea, faba- bean, bean, soybean, which were directly related to the salt induced decline in dry weight and N content of shoots. Further, faba bean and pea was severely affected by salinity while soybean was least affected indicating better adaptation of soybean nodules to salinity. Serraj et al. (1994) also reported reduced ARA and nodule respiration in soybean with NaCl salinity. Velagaleti and Schweitzer (1994) reported diversity in soybean cultivars for salt tolerance, with salt tolerant one showing better N fixation and photosynthetic efficiency, when grown in

13 nutrient media containing 7.8 dsm -1 NaCl salinity. Ikeda (1994) reported that in Trifolium repens early stage of nodulation was more sensitive to NaCl stress than the later stages; probably due to the inhibition of root hair curling. Garg et al. (1995) studied the interrelationship between the relative sensitivity, of soybean and chickpea, to salinity and the endogenous levels of cytokinins. They observed marked varibations in the two crops with soybean having higher dry weight, nitrogenase activity, nitrogen content. Similar observations have also been recorded in pigeonpea by Garg and Dua (1996). Nitrogenase activity and leghemoglobin content decreased with saline stress (100 mol m -3 NaCl) in Lupinus albus and further, nodular starch content decreased and sucrose content increased suggesting an osmotic regulation (Fernandez-Pascual,1996). Cordovilla et al. (1996) reported decline in soluble protein content of nodules and increase in proline content within nodule cytosol of Vicia faba under salinity. Further exogenous application of KNO 3 to the growth medium increased plant tolerance to salinity. Serraj and Drevon (1998) studied the interactive effects of nitrate and NaCl in alfalfa and found higher effect of NaCl on the percent N content and nitrogenase activity of N 2 fixing plants, compared to NO 3 - fed plants indicating that N 2 fixation was more sensitive to NaCl than nitrate nutrition or other functions supporting plant growth. Serraj et al. (1998) observed intraspecific variation amongst commonbean, soybean and alfalfa with common bean to be more sensitive than soybean and alfalfa under salinity in terms of nodule growth and nitrogenase activity which could be correlated to regulation of oxygen diffusion and distribution of ions in nodules. Zurayk et al. (1998) reported reduction in nodule dry weight and N-fixation in chickpea cultivars under salinity and found that symbiosis was more salt sensitive than both Rhizobium and the host plant. NaCl significantly inhibited nitrogenase Vol. 27, No. 1, activity, nodule number and dry matter accumulation per plant in all four cultivars of soybean On the basis of grafting experiments Abd-Alla et al. (1998) confirmed a shoot role in the autoregulatioin of nodule number as well as in determining salt tolerance of a genotype. Soussi et al. (1998) reported inhibition of nodulation and nitrogen fixation in Cicer arietinum cv. ILC 1919 even at lowest NaCl concentration (50 mm) while Cordovilla et al. (1999) found that nodulation and nitrogen fixation in Vicia faba was affected only by high concentrations of salinity (100 mm). Further ARA activity declined even with low salt stress in both. Soussi et al. (1999) compared the two cvs. of chickpea with differential tolerance to salinity and found the effect of salt on nodulation and nitrogen fixation more pronounced in Pedrosillano (sensitive) and reported that increase in nodular mass in ILC 1919 (tolerant) partially counteracted the inhibition of nitrogenase activity. Ferri et al. (2000) also reported inhibition in dry weight and ARA in the Phaseolus vulgaris. Nandwal et al. (2000) observed sharp decline in the leghemoglobin content and ARA of the nodules of mungbean under salinity and reported genotypic variability, with K-851 more affected than the mutant. Babber et al. (2000) reported decreased leghemoglobin, reduced ARA of nodules in chickpea plants under salinity which could be directly associated with structural changes in nodules under salt stress namely reduction in size of the nodules, decreased meristematic zone, reduced number and degradation of symbiosomes, reduced intracellular spaces and deposition of electron dense material in the intercellular spaces in the cortex of the nodules. Rao et al. (2002) studied the effect of salinity on genotypic variability amongst small seeded desi, medium seeded desi and kabuli chickpea genotypes and found kabuli genotype, CSG 8927, to be more salt tolerant with better nodulation and higher rates of nitrogen fixation than the sensitive