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Journal of Experimental Botany, Vol. 47, No. 295, pp. 203-210, February 1996 Journal of Experimental Botany Growth and nitrogen assimilation in nodules in response to nitrate levels in Vicia faba under salt stress M.P. Cordovilla, F. Ligero and C. Lluch1 Departamento de Biologfa Vegetal, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain Received 10 June 1995; Accepted 18 October 1995 Abstract This study analyses the effects of salt on the effective symbiosis of faba bean (Vicia faba L. var. minor cv. Alborea) and salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA19 grown with two KNO3 levels (2 and 8 mM). The addition of 8 mM KNO3 to the growth medium increases plant tolerance to salinity even with a concentration of 100 mM NaCI. This KN0 3 level in control plants reduced the N 2 fixation. For 2 and 8 mM KNO, the plants treated with NaCI reduced N 2 fixation to identical values. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADHGOGAT and NADH-GDH) was decreased by high KN0 3 levels. The results show that NADH-GOGAT activity was more markedly inhibited than was GS activity by salinity, therefore NADH-GOGAT limits the ammonium assimilation by nodules in V. faba under salt stress. The total proline content in the nodule was not related to salt tolerance and thus does not serve as a salttolerance index for V. faba. Key words: Glutamate synthase, glutamine synthetase, N2 fixation, nitrate, salinity. Introduction Salinity threatens irrigated agriculture in many semi-arid and arid regions of the world (Epstein, 1980; Norlyn, 1980; Staples and Toenniessen, 1984). The faba bean is considered moderately sensitive to salinity (Lauchli, 1984) and is a particularly important crop in many of these regions (World Resources, 1987). A plant's response under stress varies depending on the degree of salt stress, the stage of growth, the amount of available nutrient elements, and the type and form of the nutrient elements in the rhizosphere. Two major effects have been identified as the probable causes of salinity toxicity in various plants: the ionic effect and the osmotic effect. The ionic effect includes interference with nitrogen uptake, dislocation of nitrogen assimilation and protein assembly, interference with the transport of essential ions within the plant, and a lowering of net photosynthetic rates in the affected plants. The osmotic effect is associated with lack of cell-wall extension and cell expansion leading to cessation of growth (Downton, 1977; Huffaker and Rains, 1986). In legumes, salt stress from 50 to 200 mM NaCI significantly limits productivity by adversely affecting the growth of the host plant, the root nodule bacteria, symbiotic development and, finally, the nitrogen fixation capacity (Rai and Prasad, 1983; Bekki et al., 1987; Delgado et al., 1993). Salt stress is a major constraint in the production of legume crop species, particularly when the nitrogen needed for the growth of these plants is derived from symbiotic fixation. Plants dependent on KNO3-nitrogen are less sensitive to salt stress (Lauter et al., 1981; Singleton and Bohlool, 1984; Tu, 1981). It is generally observed that salt stress promotes the accumulation of ammonium, nitrate, and free amino acids in plants, while it reduces protein synthesis (Pessarakli et al., 1989a). Udovenko et al. (1970), investigating bean and pea plants in sand culture with various inorganic N and salt sources, showed a decreased incorporation of ammonium into amino-acid compounds by these plants. Under salt stress conditions, the non-protein-N fraction increased in peas and beans, whereas the protein-N fraction changed irregularly in stressed plants (Udovenko et al., 1970). The present study compares the effects of NaCI on ' To whom correspondence should be addressed: Fax: +34 58 2432 54. Abbreviations: ARA, acetylene reduction activity; GS, glutamine synthetase; NADH-GDH, NADH-dependent glutamate dehydrogenase; NADH-GOGAT, NADH-dependent glutamate synthase. 6 Oxford University Press 1996 204 Cordovilla et al. nitrogen-fixing V. faba plants grown with two KN0 3 levels. The aim was to assess the effect of salt on plant growth, nodulation and N2 fixation in faba bean plants grown in a solution culture containing 2 and 8 mM KNO3 and inoculated with salt-tolerant Rhizobium. The effect of salinity and the two KNO3 levels on the activity of cytosolic GS, NADH-GOGAT and NADH-GDH from nodules of salt-stressed plants was also examined. Materials and methods glutamate dehydrogenase (EC 1.4.1.2) activities were assayed spectrophotometrically at 30 °C by monitoring the oxidation of NADH at 340 nm essentially as indicated by Groat and Vance (1981) and Singh and Srivastava (1986), always within 2 h of extraction. Two controls (without a-ketoglutarate and without glutamine in the case of GOGAT, without NH^ and without a-ketoglutarate in the case of GDH) were used to correct for endogenous NADH oxidation. The decrease in absorbance (linear at least 10 min) was recorded for 8 min in a Beckman DU-70 spectrophotometer. Protein determination The soluble proteins in tissue extracts were determined by Bradford's method (Bradford, 1976), with bovine serum albumin (Merck, fraction V) as the standard. Commercial cultivar Alborea of Vicict faba L. var. minor was bought from Semillas Pacifico S.A. (Sevilla, Spain). PreContents of reduced nitrogen determination germinated seeds were planted in Leonard jars (2 per jar) with The products of acid digestion from the modified Kjeldahl vermiculite and a nutrient solution (Rigaud and Puppo, 1975), inoculated with R. leguminosarum bv. viciae strain GRA19, procedure were steam-distilled, after which N content was determined by mass spectrometry as described by Bremner which has been described as salt-tolerant (Cordovilla, 1993), (1965), and Pessarakli and Tucker (1985). and cultured in a growth chamber. Procedures and growth conditions were as described before (Cordovilla et al., 1994). Plant material and growth conditions Proline determination Salt and nitrate treatments Plants were grown on minus NaCl for 18 d from planting, after which the jars were separated into four groups. The first group continued growing on NaCl-free solution as control plants. For the second, third and fourth groups the NaCl treatrnent began on day 18 reaching the final salt concentration on day 24 (50, 100 and 200 mM NaCl, respectively). The previous assay with NaCl included two KNO3 levels, 2 and 8 mM, in each case added to the growth medium immediately after transplanting. Harvest Plants were harvested every 3d for 12 d. Harvesting started 24 d after transplanting, with 6 replicates per harvest. The plants were removed from the jars, the roots thoroughly rinsed with water, blotted dry on filter paper, and nodules picked and kept on ice. Shoot, root and nodule dry weights were recorded after 24 h at 70 CC. Six plants per treatment were used for nodule dry weight. Nitrogen fixation assays Nitrogenase (EC 1.7.99.2) activity was determined by acetylene reduction on the entire root systems of 6 plants, as well as in small nodulated root portions of the remaining plants as described by Cordovilla et al. (1994). The aliquots were analysed for ethylene in a Perkin Elmer 8600 gas chromatograph equipped with a Poropak R column (Ligero et al., 1986). Preparation of cell-free extracts and enzyme assays Trie maleic acid-KOH buffer and the extraction of nodule enzymes followed the procedure of Cordovilla et al. (1994). The supernatant obtained by 30 000 g centrifugation was assumed to be plant cell cytoplasm and used to measure enzyme activities and soluble protein. Glutamine synthetase (EC 6.3.1.2) was determined by the hydroxamate synthetase assay, adapted from Farnden and Robertson (1980) and Kaiser and Lewis (1984). Assays were optimized for the amount of enzyme to give a linear reaction within at least 30 min. Two blanks without enzyme and without L-glutamate were also analysed. NADH-glutamate synthase (EC 1.4.1.14) and NADH- Samples (1 g fresh weight) were homogenized with 10 ml of 3% (w/v) sulphosalicylic acid. The homogenate was centrifuged at 2500 g at 2°C for 10 min. The resulting supernatant was used to determine the proline content. Aliquots of 0.25 to 1 ml of crude extract were used, with the addition of 1 ml of 2.5% (w/v) ninhydrin prepared in glacial acetic acid at 60% (v/v) and phosphoric acid at 40% (v/v), 1 ml of glacial acetic acid and sulphosalicylic acid to a total volume of 3 ml. This reaction mixture is boiled for 60 min, stopped with ice for 1 or 2 min and 3 ml of toluene is added, whereupon the mixture is stirred vigorously and the upper phase is used to measure absorbance at 520 nm. A control with sulphosalicylic acid was used. To calculate the proline concentration a model curve was prepared with proline (Sigma), following the same procedure, with quantities of between 10 and 100/xg. Statistical design and analysis The experimental layout was a randomized complete block design. All values are means of 6 replicates per treatment. All results were subjected to multifactor analysis of variance with a least significant difference (LSD) test between means. Sources of variance (treatments with salt, treatments with nitrate or time) were compared with Duncan's test. Results The respective growth response to salinity stress of faba bean plants given 2 and 8 mM KNO3 can be observed in the data recorded in Table 1. These data clearly indicate that control plants (not receiving NaCl) given 8 mM KN0 3 grow more rapidly than plants given 2 mM KNO3. However, maximum dry-matter accumulation for both shoots and roots was not significantly affected by the KNO3 level. Plants fed 8 mM KNO3 are far more salt-tolerant than their counterparts fed 2 mM KN0 3 under the experimental conditions. Plants given 8 mM KN0 3 showed no reduction by salinity in the dry weight of shoots, and Nodule nitrogen metabolism 205 1 Table 1. Effect of saline treatments during the vegative growth period on dry weight in shoots, roots (g organ' ) and nodules (g plant ~l) o/V. faba plants inoculated with the salt-tolerant R. leguminosarum GRA19 and grown with 2 and 8 mM KNO3 Data in parenthesis expressed as a percentage of the control for each harvest and K N 0 3 level The least significant difference (LSD) is given for each plant organ. Plant organ NaCl (mM) KNO 3 (mM) Days after salt treatment 12 Shoot 0 2 O 0 50 0.98 1.22 1.18 1.48 0.99(84) 1.26(85) 0.97(82) 1.27(86) 0.94(80) 1.05(71) (g organ 1.42 1.68 1.26(89) 1.57(93) 1.20(85) 1.69(100) 1.15(81) 1.09(65) 2.09 2.33 1.41(67) 2.07(89) 1.21(58) 2.03(87) 1.19(57) 1.40(60) 2.22 2.28 1.82(82) 2.27(100) 1.61(73) 2.07(91) 1.43(64) 1.50(66) 0.36 0.56 0.40 0.56 0.46 0.63 0.52 0.62 0.56 0.62 0 58 0.72 0 55 0.75 0.63 0 78 0.69 0 62 0.66 0.83 0.59 1.01 0.78 0.95 0.70 0.76 0.82 0.85 0.85 1.07 0.96 0.97 0.87 0.78 0.05 0.04 0.08 0.05 0.07 0.05 0.08 0.04 0.08 0.05 (g plant" 1 ) 0.09 0.07 0.09 0.06 0.09 0.06 0.10 0.06 0.11 0.08 0.11 0.07 0.10 0.07 0.11 0.06 0.13 0.11 0.12 0.09 0.13 0.07 0.12 0.06 2 O O 100 2 200 2 Q 0 Q O LSD (0.05) 0.24 Root 0 2 0 0 50 100 200 2 8 2 8 2 0 0 LSD (0.05)0.14 Nodules 0 2 0 0 50 2 100 2 8 2 Q 0 200 LSD (0.05) 0.01 only a reduction of 34% at the 200 mM salinity level, whereas, even at a concentration of 50 mM, plants given 2 mM KNO3 showed a reduction of 18% in the dry weight of shoots, this loss increasing with the salinity level. While the plants fed 8 mM KNO3 were still growing vigorously at the 50 and 100 mM salinity levels, plants fed 2 mM were showing signs of wilting. In plants fed both KNO3 levels, the salinity effect on growth was more noticeable in the shoot than in the root. No reduction in the dry weight of the root was detected (Table 1) in any of the NaCl levels assayed. Nodule mass (Table 1) was significantly affected by KNO3, but the nodules were similar in appearance. The plants fed 2 mM KNO3 showed no reduction in drynodule mass, whereas plants fed 8 mM KN0 3 showed reduced dry-nodule mass at the end of the culture for all NaCl levels, this reduction increasing with the salinity level. The KN0 3 concentration did not significantly affect the distribution of N between roots and shoots. Furthermore, KN0 3 treatment had no effect on N content per gram (Table 2) in either shoots or roots for control plants. In salt-treated plants, the N content of shoots responded in a manner similar to that of the dry weight of shoots: the shoots of plants given 2 mM KN0 3 showed reductions at all NaCl levels assayed, and the shoots of plants given 8 mM KN0 3 showed significant reductions only at 200 mM NaCl. For roots, in both KN0 3 concentrations, all NaCl levels reduced the N content, while the dry weight remained stable. The high KNO3 (8mM) treatment in control plants caused a reduction of approximately 30% in ARA per gram of nodule and a reduction of approximately 48% in ARA per plant (Table 3). Both specific and total activity were severely depressed by salinity. The effect of NaCl on ARA was more pronounced in plants fed 2 mM KNO3, in such a way that in the last harvest the nitrogenase activity registered the same values for both KNO3 levels. Nevertheless, the evolution of both specific and total nitrogenase activity was different for the two KN0 3 concentrations, in that the plants given 8 mM KN0 3 showed the same values in all the harvests for 50 and 100 mM NaCl, while in plants given 2 mM KNO3 the decrease was progressive at all the NaCl levels. The depression in total activity was partly due to salt reducing the activity of pre-formed nodules and partly due to the reduced differentiation of new pink nodules. The activity of the enzymes mediating ammonia assim- 206 Cordovilla et al. Table 2. Effect of saline treatments during the vegetative growth period on nitrogen content of shoots and roots (mg g~l DW) faba plants inoculated with R. leguminosarum GRA19 and grown with 2 and 8 mM KNO3 of\. The LSD is given for each plant organ. Plant organ Shoot NaCl (mM) 0 50 100 200 KNO 3 (mM) Days after salt treatment 0 3 6 9 12 2 8 2 8 2 8 2 8 45.1 46.6 43.8 45.8 33.1 44.2 37.8 42.8 39.0 36.0 (mgg-'DW) 41.9 43.8 37.3 42.2 32.3 39.4 33.9 35.6 38.1 39.0 36.2 41.7 32.3 40.0 33.5 34.8 35.4 39.0 319 37.5 30.8 40.9 26.7 33.2 2 8 2 8 2 8 2 8 39.9 41.0 41.4 41.9 35.9 37.0 40.1 38.7 33.8 36.0 41.5 41.8 37.9 38.6 39.3 35.2 34.7 37.1 42.4 41.9 39.1 36.0 37.2 34.6 29.2 35.7 43.8 42.8 40.4 34.6 37.0 36.2 30.2 34.8 LSD (0.05) 4.1 Root 0 50 100 200 LSD (0.05) 1.1 Table 3 Effect of saline treatments during the vegative growth period on nodule acetylene-reduction activity (ARA) (jimol C2HA hh~lg~ 'g l ' nodule) and total ARA per plant (junol C2H4 h ' plant l) for V. faba plants inoculated with R. leguminosarum GRA19 and grown with 2 and 8 mM KN03 Parameter NaCl (mM) KNO3 (mM) Days after salt treatment 0 ARA per unit weight of nodule 0 50 100 200 2 8 2 8 2 8 2 8 83.0 59.2 LSD (0.05) 13.1 ARA per plant 0 50 100 200 2 8 2 8 2 8 2 8 4.15 2.37 3 6 9 12 (/imol C 2 H 4 h ~ ' g ~ ' nodule) 66.3 62.6 48.3 42.9 63.7 56.5 43.6 45.9 53.7 46.5 41.5 37.3 46.9 42.7 35.4 19.4 62.4 39.9 36.6 39.3 33.7 30.2 33.4 16.0 56.5 38.2 26.0 33.4 22.7 29.3 12.0 14.4 (,xmol C 2 H 4 h ~l 5.30 2.41 4.46 2.29 4.30 1.66 3.75 1.77 6.86 3.19 4.03 2.75 3.37 2.11 3.68 0.96 7.34 4.20 3.12 3.01 2.95 2.05 1.44 0.86 plant" 1 ) 5.63 3.00 5.08 2.61 4.19 2.24 4.27 1.17 LSD (0.05) 0.74 ilation in nodules was affected by different KNO3 levels (Table 4). When the nitrate supply was increased the activity of GS, NADH-GOGAT and NADH-GDH decreased. The activity of these enzymes at the last harvest was severely depressed by salinity; this reduction increased with higher levels of salinity and of KNO3, NADHGOGAT being the enzyme which showed the greatest fall in activity. After 3 d of treatment with 50 mM NaCl GS, NADH-GOGAT and NADH-GDH increased in activity at both KNO3 levels. For NADH-GOGAT and NADH-GDH an increase is also detected at 100 mM NaCl in plants grown with 2 mM KNO3. For control plants the soluble protein content (Table 5) was identical to that of plants given 2 and 8 mM KNO3. Nodule nitrogen metabolism 207 Table 4. Effect of saline treatments during the vegetative growth period on glutamine synthetase (GS) (fimol y-glutamate dehydrogenase h'1 g'1 FW), glutamate synthase (NADH-GOGAT) and glutamate dehydrogenase (NADH-GDH) (yjnol NADHOX h'^g'xFW) in nodules of V. faba plants inoculated by R. leguminosarum GRA19 and grown with 2 and 8 mN KNO3 Parameter NaCl (mM) KNO3 (mM) Days £ifter salt treatment 0 3 6 9 12 297 276 295 267 267 262 266 245 310 281 296 263 267 258 254 241 309 277 266 247 245 226 228 206 lTl 120 156 127 142 117 111 107 136 120 135 96 116 94 91 79 130 112 129 89 108 88 69 67 112 97 111 91 86 64 49 46 31.5 29.2 34.7 32.8 34.3 28.5 30.1 28.2 32.9 31.2 30.5 26.9 29.7 26.2 27.5 25.0 30.0 27.4 29.6 26.4 29.4 23.5 24.1 22.8 30.4 25.3 29.4 23.3 29.0 23.1 22.3 17.6 (,dnol y-glutamyl-hydroxamate h"'g" 1 FW) GS 0 50 100 200 2 8 2 8 2 8 2 8 302 283 296 284 307 301 299 270 300 277 LSD (0.05) 7 NADH-GOGAT 0 50 100 200 LSD (0.05) 2 NADH-GDH 0 50 100 200 2 8 2 8 2 8 2 8 2 8 2 (/imol NADH^h-V1 FW) 130 127 32.8 26.5 8 2 8 2 8 LSD (0.05) 1.3 (Hafeez et al., 1988). Similarly in this work, V. faba cv. Alborea plants given 2 mM KNO3 and exposed to 50 mM NaCl decrease the shoot dry weight per plant (Table 1). However, plants fed 8 mM KNO3 showed no reduction in shoot dry weight when exposed to 50 mM and 100 mM NaCl, although this parameter was reduced by 200 mM NaCl. Therefore, in these experiments, plants given 8 mM KNO3 were more salt tolerant than were plants fed 2 mM KNO3. These responses to salinity are generally consistent with conclusions that N-fixing plants are more sensitive to salinity than N-fertilized plants (Lauter et al., 1981; Alston and Graham, 1982; Yousef and Sprent, 1983). Torres and Bingham (1973) suggest that NOf deficiency induced by Cl" as a result of antagonism between Discussion ions, retards growth in plants exposed to high NaCl levels. It is conceivable that the addition of NO3" decreases Growth and dry-matter accumulation of legumes are the Cl~ level in plant tissues (Feigin et al., 1984). reportedly reduced by low salinity levels (about 50 mM Silberbush and Lips (1988) as well as Martinez and Cerda NaCl) in Vicia faba and Phaseolus vulgaris (Abdel(1989) demonstrate that NOf in solution decreases the Ghaffar et ai, 1982), Glycine wightii (Wilson, 1970), Glycine max (Grattan and Maas, 1988), and Vigna radiata accumulation of Cl~, but does not affect the Na + content. After 12 d of saline treatment at all the NaCl levels, the soluble protein content of the nodule decreased more in plants with 2 mM KNO3. This reduction increased with higher NaCl concentrations in the medium. The soluble proline content in the nodule (Table 5) was higher in plants given 8 mM KNO3. The response to NaCl varied with the NaCl level and KN0 3 level. In the plants given 2 mM KNO3 the proline content did not change with 50 mM NaCl, whereas with 100 and 200 mM NaCl the proline content rose 20 and 10 times, respectively, with regard to the first harvest. Similarly, plants given 8 mM KN0 3 showed 10-fold proline increases at all the NaCl concentrations assayed. 208 Cordovilla et al. Table 5. Effect of saline treatments during the vegetative growth period on soluble protein concentration (mg g~l FW) and proline content (pmol g~yFW) in nodules 0/V. faba plants inoculated by R. leguminosarum GRA19 and grown with 2 and 8 mN KNO3 Parameter Protein NaCl (mM) 0 50 100 200 KNO 3 (mM) 2 8 2 8 2 8 2 8 Days after salt treatment 0 3 6 9 12 13.3 13.1 13.3 13.6 13.2 13.4 12.4 12.4 12.7 12.9 (mgg-'FW) 13.2 13.3 13.0 12.7 12.4 12.1 12.1 12.3 13.7 13.0 11.8 12.8 11.7 12.4 11.1 12.4 13.1 13.2 11.6 12.5 11.3 12.5 10.7 11.9 0.16 0.40 0.17 0.48 0.17 0.63 0.31 0.84 (mgg-'FW) 0.16 0.52 0.19 0.96 0.66 1.49 1.44 2.17 0.18 0.56 0.23 1.03 2.33 2.31 3.09 5.12 0.18 0.55 0.25 4.31 2.69 6.30 3.09 8.03 Macroptilium, Neonotonia, LSD (0.05) 0.5 Proline 0 50 100 200 2 8 2 8 2 8 2 8 0.14 0.30 LSD (0.05) 0.25 This may explain the slight inhibition of growth for concentrations of 100 mM NaCl in V. faba plants grown with high KNO3 levels. Salinity affected shoot growth more than root growth, as was also reported for beans (Wignarajah, 1990). In these experiments, shoot N content responded in a manner comparable to that of growth. However, in the root, N content was decreased by salinity contrary to growth, as noted in other legumes (Hafeez et al., 1988; PessarakJi et al., 19896). Other authors observed no reduction in N content (Singleton, 1983; Weil and Khalil, 1986). In this research, with greater KN0 3 dosages, salinity had a less inhibitory effect on the N content in V. faba cv. Alborea—results which agree with those of Yousef and Sprent (1983) with other V. faba cvs administered NH4NO3. The 8 mM KNO3 treatment affected the faba bean nodule by depressing nodule mass, as opposed to the findings of Caba et al. (1990). Salt stress, together with the high KN0 3 level decreases nodule mass, undetected in plants grown with 2 mM KNO3. In fact, nodulation and nitrogenase activity correlate negatively with the inorganic nitrogen concentration in the soil (Alston and Graham, 1982). In control plants, the ARA was affected by high KNO3 concentrations (30%). In field experiments, other authors have reported that V. faba plants can prefer N2, with an apparent nitrate tolerance (Hardanson et al., 1991). A notable decline in ARA occurred with low-level salt stress in plants given 2mM KN0 3 . This finding corroborates earlier observations concerning Glycine, Medicago, and Phaseolus (Berstein and Ogata, 1966; Lakshmi et al., 1974; AbdelGhaffar et al., 1982; Wilson, 1985), and more recent findings in Vigna radiata (Hafeez et al., 1988), Cicer arietinum (Elsheikh and Wood, 1990) and Arachis hypogea (Leidi et al., 1992). Low level NaCl plant growth did not change; this decline in ARA may be attributable to a direct effect of salt on nitrogenase. This conclusion agrees with Burns et al. (1985), who reported that NaCl directly affected nitrogenase purified from Azotobacter. The present study shows that in nodules of V. faba cv. Alborea nitrogen nutrition interferes with the activity of the enzymes mediating assimilation of nitrogen. In control plants, GS activity was approximately 2.4 times higher than NADH-GOGAT activity, for the two levels of KN0 3 assayed. The GDH activity was roughly 10 times and 4 times less than GS and GOGAT values, respectively, for the two KNO3 levels. Therefore, NH3 is assimilated via the GS/GOGAT system in the nodule cytosol of V. faba cv. Alborea. These results correspond with those for V. faba (Caba et al, 1993) and for alfalfa (Ta et al., 1986). Groat and Vance (1981) observed that in Medicago sativa the GDH activity is not associated with nitrogen fixation either, but rather is related with nodule senescense. Salt stress inhibited GS and NADH-GOGAT activities, a finding in agreement with Bourgeais-Chaillou et al. (1992), who reported reduced GS and NADH-GOGAT in the soybean. Treatment with NaCl in V. faba cv. Nodule nitrogen metabolism Alborea affected NADH-GOGAT more than GS, as reported by Billard and Boucaud (1980) for Phaseolus vulgaris. In the first 3 d salt treatment stimulated (at certain NaCl concentrations only) the activity of enzymes involved in ammomium metabolism. This stimulation may be due to the NH^ and amide accumulation induced by stress (Hatata, 1982). The decrease in soluble protein content of the nodules is a common response to salt stress reported in other legumes (Bourgeais-Chaillou et al., 1992). The response may be due to a protein break-down (Mothes, 1956), or to an alteration in the incorporation of amino acids into proteins (Stewart and Lee, 1979). Udovenko et al. (1970) states that salt stress reduces amino acid incorporation into proteins in V. faba and P. sativum. The effect of salt on soluble protein in the nodule is less when plants are grown with high KN0 3 concentrations. The proline content within the cytosol of the nodule of V. faba cv. Alborea increased under salt stress, as also described for other legumes (Kohl et al., 1991). At the end of culture, the increase in proline, with respect to plant growth without salt, for 100 mM (15- and 12-fold for 2 and 8 mM KNO3, respectively) and 200 mM NaCl (17- and 15-fold for 2 and 8 mM KNO3, respectively) (Table 5) was similar to that described by Fougere et al. (1991) for nodules of soybean plants grown in the presence of 150 mM NaCl. Marked increase (10-fold or more) in free proline occurs in many plants during moderate or severe water or salt stress; this accumulation, mainly as a result of increased proline biosynthesis, is usually the most outstanding change among the free amino acids (Hanson and Hitz, 1982). Therefore, as reported in roots, stems, and leaves of other plants, proline accumulation in nodules may represent an osmoregulatory mechanism. There is great controversy over proline accumulation, which appears to be more a symptom of susceptibility to stress (Hanson and Hitz, 1982) than an adaptive response. Plants grown with 8 mM KN0 3 reach higher proline levels than do plants with 2 mM KNO3. However, the proline level did not always correlate with the ability to withstand salinity stress. Plants with 8 mM KNO3 and 200 mM NaCl, and plants with 2 mM KN0 3 for all levels of NaCl showed strong inhibition of growth and significant increases in proline (Tables 1, 2). In these cases, part of the proline could result from catabolic processes that accompany a decrease in the growth rate. Thus, in V. faba cv. Alborea, the proline content of the nodule is not a reliable index of salt tolerance, as shown for proline accumulation in leaves of Vigna (Ashraf, 1989). In conclusion, V. faba cv. Alborea plants given 8 mM KNO3 tolerate 100 mM NaCl, and an increase in proline content was noted, correlating with the rise in salt, but this increase might not be sufficient to confer resistance in the cultivar used. 209 Acknowledgements Financial support was obtained through the Andalusian Research Program and the DGICYT. References Abdel-Ghaffar AS, El-Attar HA, El-Halfawi MH, Abdel-Salam AA. 1982. Effect of inoculation, nitrogen fertilizer, salinity and water stress on symbiotic N2-fixation by Vicia faba and Phaseolus vulgaris. In: Graham PH, Harris SC, eds. Biological nitrogen fixation technology for tropical agriculture. Colombia: Centro International de Agriculture Tropical de Cali, 153-60. Alston AM, Graham RD. 1982. The influence of soil nitrogen status and previous crop on nitrogen fixation (acetylene reduction) in barrel medic, Medicago trunculata Gaertn. Australian Journal of Soil Science 27, 462-9. Ashraf M. 1989. 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