CHANGES IN THE NODULAR METABOLISM IN DESI AND KABULI GENOTYPES OF CHICKPEA (CICER ARIETINUM L.) UNDER SALT STRESS

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1 Legume Res., 29 (1) : 1-10, 2006 CHANGES IN THE NODULAR METABOLISM IN DESI AND KABULI GENOTYPES OF CHICKPEA (CICER ARIETINUM L.) UNDER SALT STRESS Neera Garg and Ranju Singla Department of Botany, Punjab University, Chandigarh , India ABSTRACT Experiments were conducted to study the nodular metabolism, in order to draw comparisons between nitrogen fixation, carbon fixation and ammonia assimilation in nodule cytosol of desi (CSG 8962, DCP 92-3) and kabuli (CSG 9651, BG 267) genotypes of chickpea. Maximum acetylene reduction activity (ARA) was observed in CSG 9651 and minimum in DCP 92-3.The specific activity of ammonia assimilating enzymes GS, GDH, GOGAT was less affected in the CSG 9651, CSG 8962 as compared to BG 267, DCP GS activity was more tolerant of salinity than GOGAT and it limits ammonia assimilation under saline stress. Nodule phosphoenol pyruvate carboxylase (PEPC) activity was enhanced by salt stress and was clearly related to salt concentration. Chickpea genotypes tolerant to salt stress have better enzyme activities responsible for higher nodule nitrogen fixation. INTRODUCTION Legumes belonging to family Fabaceae, the third largest family of angiosperms with three sub families comprising 650 genera and more than 18,000 species (Polhill, 1994) rank next to cereals in importance as food and are valued for their ability in fixing atmospheric dinitrogen. Leguminous plants, rather root nodules are by far the largest sole source of organic nitrogen in global nitrogen cycle. Soil salinity limits the production of grain legumes, inhibiting the establishment of legume-rhizobia symbiosis and ultimately nitrogen fixation (Serraj et al., 1998). 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. Detrimental effects of salts on plants are either due to toxicity of specific ions or osmotic effects. Osmotic effects imposed water stress eventually lower the growth while ionic effects interferes with nitrogen fixation, assimilation and lower the net photosynthetic rate (Cordovilla et al.,1996). Chickpea (Cicer arietinum) is an important pulse crop in semi arid and arid areas and its production is severely affected due to salinity (Ashraf and Waheed, 1993). Cultivars grown in India are either desi (native) types or kabuli (Mediterranean) types. Although these both types are at par in terms of food quality, kabuli types have been reported to have relatively higher salt tolerance towards salinity than the desi (Dua and Sharma, 1995; Rao et al., 2002). The present study was directed to compare variability of salinity resistance between the desi and kabuli genotypes of chickpea for efficient nitrogen, carbon fixation and ammonia assimilation as well as to identify important metabolic markers responsible for salt resistance. MATERIAL AND METHODS The seeds of chickpea cultivars were obtained from Central Soil Salinity Research Institute (CSSRI) Karnal and were inoculated with salt tolerant Mesorhizobium ciceri strain F: 75 procured from IARI, New Delhi, India. Preliminary experiments were conducted on the effects of salt stress on growth and yield in

2 2 LEGUME RESEARCH ten different cultivars of chickpea. On the basis of the above screening, four genotypes, two each of desi (DCP 92-3 and CSG 8962) and two kabuli (BG 267 and CSG 9651) differing in their salt sensitivities i.e. former most salt sensitive and latter most salt tolerant respectively, amongst the two groups, were selected for a detailed study. Seeds were surface sterilized in 30 per cent (w/v) mercuric chloride for 2 min, then washed with sterile water and germinated in earthenware pots lined with polythene bags. The pots were treated with a mixture of sodium chloride, calcium chloride and sodium sulphate of various concentrations i.e. 0, 4, 6, 8 dsm -1 electrical conductivity from 15 days of sowing till harvest time. Three plants of uniform size were maintained in each pot. Plants were sampled and analyzed for following parameters at 40, 70, 100 DAS. Two pots with three plants each were sampled per treatment. The plant samples were dried in an oven for 72 h for 70 0 C to record dry weights of nodules. Leghemoglobin content in nodules was determined according to Hartree (1957). The nodules (0.5 g) were homogenized in 5 ml extraction medium containing 0.05 M phosphate buffer and the precipitate of the nodular extract was obtained by raising the ammonium sulphate saturation percentage. Absorbance was read at 545nm and plotted against the graded concentrations of hemin. Nitrogenase activity in root nodules was determined by the method of Herdina and Silsbury (1990). The aliquots were analyzed for ethylene reduction in Perkin Elmer 8600 gas chromatograph equipped with Porapak R column (Ligero et al., 1986). Nitrate reductase activity was assayed in leaf tissue according to method of Nair and Abrol (1973). The absorbance of developed colour was read at 540 nm and the enzyme activity was expressed as µ mol NO 3 reduced g- fr. wt. h -1 Ammonia assimilating enzymes nodules sample (0.5-1 g fr. wt.) were weighed and homogenized in a chilled mortar with pestle using 5 ml 0.1 M phosphate buffer (ph 6.8). The homogenate was centrifuged at 10,000 rpm for 20 min at 0 degree C. The clear supernatant was assayed and used as crude extract for enzyme activity of glutamate dehydrogenase. For glutamate synthase assay, extraction medium used was 5 ml of phosphate buffer (ph 7.2) containing 1 mm disodium EDTA, 1mM dithioerythriol and 1% polyvinyl pyrrolidone. For glutamine synthetase assay, the extraction medium used was 5 ml ice cold 0.1M tris HCl buffer (ph 7.6). Glutamate synthase and Glutamate dehydrogenase activities were assayed spectrophotometrically by monitoring the oxidation of NADH at 340nm essentially as indicated by Tempest et al. (1970) and Pahlich and Joy (1971). Two controls (without alpha ketogluatrate} were used to correct for endogenous NADH oxidation. Glutamine synthetase was assayed according to Rowe et al. (1970). Two controls (without ATP and sodium glutamate) were also analyzed. The absorbance of supernatant was recorded at 535nm against reagent blank. In vitro phosophenol pyruvate carboxylase (PEPC) activity was assayed by the method of Christellar et al. (1977). Nodule extract was prepared in ice cold extraction medium containing 50mM tris HCl, 10mM MgCl 2 and 5mM DTTwith ph adjusted to 8.0.The homogenate was centrifuged at 35, 000 x g for 20 min and supernatant obtained was assayed according to procedure of Maruyama et al. (1966). In vivo phosophenol pyruvate carboxylase (PEPC) activity was estimated by exposing nodules to 14 CO 2 in a plexiglass chamber using the method of Kar et al. (1990).The data was statistically analyzed following the method of analysis of variance. All the values are means of six replicates.

3 Vol. 29, No. 1, RESULTS AND DISCUSSION Acetylene reduction activity (ARA) of nodules decreased consistently in all cultivars under salinity and the adverse effects became accentuated as duration of stress increased (Table 1). Cultivars CSG 9651 and CSG 8962 showed significantly higher nitrogenase activity as compared to the BG 267 and DCP Sensitive cultivars showed lower ARA values than the tolerant ones with salt treatment. Decreased ability of nodules to reduce C 2 H 2 under salinity has been well documented in other legumes (Serraj et al., 1998; Ferri et al., 2000). According to Serraj et al. (2001), the short term inhibition of ARA by salt stress may be due to nodule oxygen limitation by oxygen diffusion or due to toxic effects of Na or Cl accumulation. The inhibition of ARA by the salt stress in the present study seems to be due to the toxic effects of Na ions. Lb content of nodules was also affected by saline stress and showed significant damage with increasing concentrations of salts in all the cultivars (Fig 1). The damage was of much lesser degree in CSG 9651 and CSG 8962, while it was of higher degree in BG 267 Table 1. Effect of different levels of salinity on the rate of nitrogenase activity (n. moles ethylene/mg nodule dry weight/h) in the desi and kabuli cultivars of chickpea {figures in parenthesis represent per cent decrease (-) over control} Days after sowing Electrical conductivity (dsm -1 ) (DAS) Control CSG ± ± ± ± (-02.9) (-06.6) (-11.4) ± ± ± ± (-03.9) (-07.1) (-12.6) ± ± ± ± (-04.8) (-10.8) (-18.0) BG ± ± ± ± (-15.7) (-22.3) (-27.6) ± ± ± ± (-21.3) (-33.4) ± ± ± ± (-27.1) (-33.8) (-47.4) CSG ± ± ± ± (-07.4) (-12.7) (-15.9) ± ± ± ± (-08.1) (-13.5) (-19.8) ± ± ± ± (-11.2) (-16.9) (-22.5) DCP ± ± ± ± (-21.5) (-27.4) (-37.2) ± ± ± ± (-23.0) (-32.3) (-40.0) ± ± ± ± (-32.5) (-46.5) (-60.4) Values are means ± SE of six replicates.

4 4 LEGUME RESEARCH μg Leghemoglobin per g nodule fresh weight 40 DAS 70 DAS 100 DAS Electrical conductivity dsm -1 Fig. 1. Effect of different levels of salinity on the Leghemoglobin content (μg/g fresh weight) in the desi and kabuli cultivars of chickpea (Mean of six replicates) Glutamine synthetase (GS) units per mg protein 40 DAS 70 DAS 100 DAS Electrical conductivity dsm -1 Fig. 2. Effect of different levels of salinity on the Glutamine synthetase (GS) activity (units/mg protein) in the nodules in desi and kabuli cultivars of chickpea (Mean of six replicates)

5 Vol. 29, No. 1, Glutamine synthetase (units per mg protein) 40 DAS 70 DAS 100 DAS Electrical conductivity dsm -1 Fig. 3. Effect of different levels of salinity on the Glutamine synthetase (GOGAT) activity (units per mg protein) in the nodules in desi and kabuli cultivars of chickpea (Mean of six replicates) Glutamate dehydrogenase (GDH) units per mg protein 40 DAS 70 DAS 100 DAS Electrical conductivity dsm -1 Fig. 4. Effect of different levels of salinity on the Glutamate dehydrogenase (GDH) activity (units per mg protein) in the nodules in desi and kabuli cultivars of chickpea (Mean of six replicates)

6 6 LEGUME RESEARCH and DCP The kabuli CSG 9651 had maximum Lb content, followed by CSG 8962, BG 267 and DCP 92-3 under all the salinity treatments and at all the sampling stages. Since Lb plays an important role in facilitating diffusion of oxygen to the bacteroids, a decrease in its content might contribute to increased resistance to diffusion of oxygen inside the nodules and thus adversely affect the nitrogen fixation (Aquirreolea and Sanchez- Diaz, 1989). The drop in Lb content is correlated to the loss of C 2 H 2 reduction activity (Delgado et al., 1994).These findings may be directly related to the maintenance of C 2 H 2 reduction capacity and therefore an adaptation of the nodules of tolerant chickpea cultivars to salinity. The enzyme activities related to the nitrogen assimilation i.e. glutamate synthetase, glutamine synthase and glutamate dehydrogenase were measured in the nodule cytosol and the data (Figure 2, 3, 4) showed GS and GOGAT activity to be significantly higher as compared to GDH. Salinity inhibited all these activities and this inhibition increased with increase in salt concentration and with time. Out of four cultivars, maximum degradation in enzyme activities was observed in DCP 92-3 indicating it to be highly salt sensitive while CSG 9651 proved to be salt Table 2. Effect of different levels of salinity on the nitrate reductase (NR) activity (µ mol NO 2 /g FW/hr) in roots in the desi and kabuli cultivars of chickpea {figures in parenthesis represent percent decrease (-) over control} Days after sowing Electrical conductivity (dsm -1 ) (DAS) Control CSG ± ± ± ± (-4.2) (-7.3) (-11.5) ± ± ± ± (-6.7) (-10.9) (-13.2) ± ± ± ± (-8.1) (-12.8) (-15.8) BG ± ± ± ± (-13.4) (-17.8) (-20.3) ± ± ± ± (-15.3) (-19.7) (-24.1) ± ± ± ± (-20.1) (-26.7) (-30.2) CSG ± ± ± ± (-8.6) (-10.1) (-13.7) ± ± ± ± (-11.4) (-15.0) (-17.3) ± ± ± ± (-13.3) (-18.1) (-21.1) DCP ± ± ± ± (-16.6) (-21.6) (-30.4) ± ± ± ± (-20.7) (-28.5) (-35.9) ± ± ± ± (-28.9) (-35.9) (-40.1) Values are means ± SE of six replicates.

7 Vol. 29, No. 1, resistant, showed higher enzyme activity at all salinity levels. Further, GOGAT activity was more limited by salinity as compared to GS activity. The studies indicated that GS/GOGAT cycle rather than GDH was responsible for ammonia assimilation in the nodules of chickpea cultivars and salt stress interferes with the activity of these enzymes. These results are in line with those of Cordovilla (1993); Cordovilla et al. (1996), though stimulation in enzyme activities was reported by Soussi et al. (1998). Our findings indicated that NADH- GOGAT activity was more markedly inhibited than GS activity, suggesting that latter enzyme is tolerant to salt stress. It also suggested that GDH activity is not associated with nitrogen fixation as indicated by Groat and Vance (1981). GS activity was on an average higher than GOGAT activity as earlier reported by Cordovilla et al. (1999). Nitrate reductase activity was analyzed so as to have a comparative analysis of both the enzymes i.e. NR and nitrogenase under salt stress in kabuli and desi cultivars of chickpea (Table 2). The enzyme activity was high at 40 DAS, reached its peak at 70 DAS and declined thereafter towards 100 DAS. CSG 9651, CSG 8962 showed higher NR activity as compared to BG 267, DCP 92-3 where the NRA was relatively low. These Table 3. Effect of different levels of salinity on the in vitro PEPC activity ( 14 CO 2 fixed 10 3 DPM/g/h) in the nodules in the desi and kabuli cultivars of chickpea {figures in parenthesis represent per cent increase (+) or decrease (-) over control} Days after sowing Electrical conductivity (dsm -1 ) (DAS) Control CSG ± ± ± ± (+14.1) (+21.9) (+26.7) ± ± ± ± (+21.6) (+30.2) (+38.3) ± ± ± ± (+29.1) (+39.6) (+50.5) BG ± ± ± ± (+6.0) (-10.1) (-14.7) ± ± ± ± (+8.3) (-15.6) (-24.8) ± ± ± ± (+12.2) (-23.6) (-31.2) CSG ± ± ± ± (+8.5) (+14.2) (+20.4) ± ± ± ± (+14.0) (+23.2) (+27.3) ± ± ± ± (+22.4) (+30.4) (+39.0) DCP ± ± ± ± (+3.8) (-11.0) (-20.4) ± ± ± ± (+5.9) (-21.2) (-27.5) ± ± ± ± (+9.8) (-29.4) (-38.3) Values are means ± SE of six replicates.

8 8 LEGUME RESEARCH Table 4. Effect of different levels of salinity on the in vivo PEPC activity ( 14 CO 2 fixed 10 3 DPM/g/h) in the nodules in the desi and kabuli cultivars of chickpea {figures in parenthesis represent per cent increase (+) or decrease (-) over control} Days after sowing Electrical conductivity (dsm -1 ) (DAS) Control CSG ± ± ± ±0.004 (+12.1) (+19.8) (+25.1) ± ± ± ±0.003 (+23.6) (+30.5) (+40.1) ± ± ± ±0.006 (+33.1) (+41.9) (+56.8) BG ± ± ± ±0.007 (+5.2) (-9.1) (-12.3) ± ± ± ±0.004 (+8.7) (-12.6) (-20.3) ± ± ± ±.001 (+10.3) (-23.5) (-30.7) CSG ± ± ± ±0.007 (+9.6) (+15.7) (+21.9) ± ± ± ±0.006 (+20.5) (+28.5) (+35.8) ± ± ± ±0.006 (+28.5) (+36.0) (+44.5) DCP ± ± ± ± (+3.9) (-11.2) (-16.8) ± ± ± ±0.007 (+5.6) (-17.5) (-25.1) ± ± ± ±0.001 (+8.4) (-26.0) (-39.0) Values are means ± SE of six replicates. observations are in agreement with other findings, indicating a similar inhibition in NRA due to NaCl (Goula et al., 1994; Khan, 1996). According to Solomonson and Barber (1990), under salt stressed conditions, the alteration of function, such as enzyme synthesis, rate of destruction or specific activity change, the rate of production of end product might be responsible for decreased NRA. Kabuli cultivar CSG 9651 had the highest NR activity which might be responsible for the higher ammonia assimilation leading to higher synthesis of amino acids. Carbon dioxide fixation in the nodules in closely associated with nitrogen fixation. The present study showed that PEPC activity was stimulated in response to salt stress, the quantum of stimulation being concentration dependent. However, in sensitive cultivar DCP 92-3 the activity increased under lower saline concentrations but declined with higher salt dosages (Table 3, 4). Delgado et al. (1993), Soussi et al. (1998, 1999) have also reported an increase in nodules PEPC activity under salt stress. Guerrier (1988) has proposed that higher PEPC activity could be used as a

9 Vol. 29, No. 1, biochemical indicator of salt tolerance. Drevon et al. (1998) has suggested that PEPC may be involved in the regulation of turgescence or active osmocontraction of cells of the inner cortex, which is a proposed mechanism of oxygen diffusion barrier. The higher PEPC activity in the tolerant cultivars could improve regulation of O 2 diffusion. The increase in specific PEPC activity in stressed nodules is not only related to the reduction in protein concentration but is also due to an increase in total PEPC activity (Delgado et al., 1993). Further the lack of photosynthate did not inhibit PEPC- MDH pathway which supports the hypothesis concerning the limitation in supply of energy substrate mainly malate to the bacteroids. The inhibition of nitrogenase activity by salt stress may be a consequence of the decrease in malate content in the nodules (Soussi et al., 1999). A decrease in Lb content, respiration capacity of the bacteroids and malate concentration in the nodules, as induced by salt stress, could be some important mechanisms involved in the inhibitory effect of salinity on nitrogen fixation. In the light of present results, discussed in details as above, it may be concluded that saline soils inhibit symbiotic performance of different genotypes of chickpea. However, important variability in terms of nitrogen assimilation and carbon metabolism were observed amongst different genotypes of chickpea. The greater performance of symbiosis under saline conditions seems to be determined mainly by the tolerance of the legume host plant. In general, both kabuli genotypes seemed to have a better potential for salt tolerance as compared to the desi. Even the sensitive kabuli exhibited significantly higher salt resistance than the sensitive desi. Tolerance to salinity seemed to be directly related to a number of physiological and biochemical traits such as stimulation in nodule PEPC activity, higher leghemoglobin, nitrogenase, NR and ammonia assimilating enzyme activities in the tolerant kabuli and desi genotypes of chickpea. These parameters proved to be excellent indices of tolerance to saline stress in this species. Changes in the nodular metabolism as indicated by salt stress seemed to be directly related to the relative salt tolerance or susceptibility of chickpea genotypes. REFERENCES Aquirreolea, J. and Sanchez-Diaz, M. (1989). J. Pl. Physiol., 134: Ashraf, M. and Waheed, A. (1993). Pl. Soil., 154: Christellar, J.T. et al. (1977). Pl. Physiol., 60: Cordovilla, M.P. (1993). Ph.D. Thesis, University of Granada. Cordovilla, M.P. et al. (1996). J. Expt. Bot., 47: Cordovilla, M.P. et al. (1999). Applied Soil Eco., 11: 1-7. Delgado, M.J. et al. (1993). Physiol. Plant, 89: Delgado, M.J. et al. (1994). Soil Biol. Biochem., 26: Drevon, J.J. et al. (1998). Biological Nitrogen Fixation for the 21 st Century. Kluwer Academic Publishers, Dordrecht, Dua, R.P. and Sharma, P.C. (1995). ICPN, 2: Ferri, A. et al. (2000). Pl. Biol., 2: Goula, H. et al. (1994). Pl. Physiol., 105: Groat, R.G. and Vance, C.P. (1981). Pl. Physiol., 67: Guerrier, G. (1988). Seed Sci. Tech., 16: Hartree, E.F. (1957). Modern Methods of Plant Analysis. (Paech, K. and Tracey, M.V. eds.) (Springer-Verlag) pp Herdina and Silsbury, J.H. (1990). Aust. J. Pl. Physiol., 17: Kar, M.; Mythili, J.B. and Nair, T.V.R. (1990). J. Nuclear Agric. Biol., 19: Khan, M.G. (1996). Indian J. Pl. Physiol., 1(2):

10 10 LEGUME RESEARCH Ligero, F. et al. (1986). J. Pl. Physiol., 125: Maruyama, H.I. et al. (1986). J. Biochem., 241: Nair, T.V.R. and Abrol, Y.P. (1977). Crop Sci., Pahlich, E. and Joy, K.W. (1971). Can. J. Biochem., 49: Polhill, R.M. (1994). In: Phytochemical Dictionary of Leguminosae, XXXV-XXVIII. Chapman and Hall, New York, NY. Rao. D.L.N. et al. (2002). Annals Bot., 89: Rowe, W.B. et al. (1970). In: Methods in Enzymol. Vol 17 Part A (Tabor, H. and Tabor, W. eds.). pp Serraj, R. et al. (2001). Agronomie, 21: Serraj, R. et al. (1998). J. Plant Nutr., 21: Solomonson, L.P. and Barber, M.J. (1990). Annu. Rev. Pl. Physiol. Pl. Mol. Biol., 41: Soussi, M. et al. (1998). J. Expt. Bot., 49(325): Soussi, M. et al. (1999). J. Expt. Bot., 50(340): Tempest, D.W. et al. (1970). Biochem. J., 117:

GROWTH AND NODULATION OF MUNGBEAN (VIGNA RADIATA [L.] WILCZEK) AS AFFECTED BY SODIUM CHLORIDE

Journal of Research (Science), Bahauddin Zakariya University, Multan, Pakistan. Vol.15, No.2, June 2004, pp. 139-143 ISSN 1021-1012 GROWTH AND NODULATION OF MUNGBEAN (VIGNA RADIATA [L.] WILCZEK) AS AFFECTED