This work was financed by proyect AGL 2008-00155/AGR

1. M. truncatula var. Jemalong M. truncatula var. Jemalong M. truncatula var. Jemalong M. truncatula var. R-108 M. truncatula var. R-108 M. truncatula var. R-108 PDW NFR g plant-1 µmol N2 g-1 NDW h-1 Figure 1. Effect of the NaCl and mannitol treatments on plant dry weight (PDW) and nitrogen fixation rate (NFR) in two varieties ofMedicago truncatula with differential salt tolerance, M. truncatula var. Jemalong (salt tolerance) and M. truncatula var. R-108 (salt sensitive) inoculated with Sinorhizobium meliloti GR4. GS GOGAT GDH Myoinositol Pinitol Mannitol mg-1 g-1 FW mg-1 g-1 FW nmol NADH mg-1 prot min-1 nmol NADH mg-1 prot min-1 mg-1 g-1 FW nmol γ-glutamil mg-1 prot min-1 Figure 2. Response of enzymes responsible for NH4+ assimilation, Glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate deshydrogenase (GDH) activities, in nodules of two varieties ofMedicago truncatula with differential salt tolerance, M. truncatula var. Jemalong (salt tolerance) and M. truncatula var. R-108 (salt sensitive) under NaCl and mannitol stress. Figure 3. Content of myoinositol, pinitol and mannitol in nodules of two varieties ofMedicago truncatula with differential salt tolerance, M. truncatula var. Jemalong (salt tolerance) and M. truncatula var. R-108 (salt sensitive) under NaCl and mannitol stress. Comparative responses of twovarieties of Medicagotruncatulatosalinity and osmotic stress José A. Herrera-Cervera, Néstor Fernández del Saz and Francisco J. Palma Martín Department of PlantPhysiology, Faculty of Science, University of Granada, (18071) Granada, Spain INTRODUCTION Biological nitrogen fixation represents the major source of nitrogen input to agricultural land, including arid regions. Symbiotic systems play a significant role in this process by improving fertility and productivity of nitrogen-poor soils. However, Rhizobium-legume symbiosis is affected by different environmental factors, such as salt and osmotic stress. The negative effects of NaCl are due mainly to the accumulation of toxic levels of ions and reduced availability of water due to osmotic stress. The stresses suffered by plants under salinity include osmotic stress and specific ion stress (Munns and Tester, 2008), the osmotic effect involves limited water absorption due to salinity in the rhizosphere and the ionic effect consists of intracellular toxicity or imbalance due to excess ions (Zhu, 2001). Since both osmotic and ionic damages are interrelated and co-exist under saline conditions, separating the two components is an important step in understanding the basis of tolerance. The polyethylene glycol and mannitol applied to the nutrient solution is used as a means to induce water stress in crop plants and tissues, however, mannitol is considered a compatible solute, which can accumulate to high concentrations in plant cells without affecting cell metabolism. We studied the effect of NaCl and iso-osmotic nutrient solutions with mannitol, on two varieties of Medicagotruncatula with differential salt tolerance, M. truncatula var. Jemalong (salt tolerance) and M. truncatulavar R-108 (salt sensitive) with the aim of distinguish between the osmotic and specific ionic effects. We have focused, among others, on growth and nitrogen fixation rates, ammonium metabolism and polyols. MATERIALS AND METHODS *Biological material and growthconditions: Plants of Medicagotruncatulavar. Jemalong (salt tolerance) and var. R-108 (salt sensitive) inoculated with SinorhizobiummelilotiGR4, were grown in a controlled environmental chamber. When plants were 35 days old (symbiosis established) were treated with sodium chloride and mannitol (0, 50, 100 mM) added to the growth medium. Plants were harvested 56 days after sowing. *Nitrogenfixationratewas measured as the representative H2 evolution in an open-flow system (Witty and Minchin, 1998). *Enzyme activities glutamine synthetase (GS; Wallsgrove et al., 1979), glutamate synthase (GOGAT; Groat and Vance, 1981) and glutamate dehydrogenase (GDH; Singh and Srivastava, 1986). *Myoinositol, pinitol and mannitol were separated and quantified by isocratic ion chromatography with pulsed amperometric detection according to (Cataldi et al., 2000) with modifications, conducted on a Dionex ICS-3000 chromatograph (Dionex Corp. Sunnyvale, California). RESULTS AND DISCUSSION To investigate the effects of exogenous NaCl and mannitol in Medicago truncatula, we measured plant dry weight (PDW). We found that salt and mannitol stress reduced PDW in both varieties (Figure 1), although in M. truncatula var. R108 (salt sensitive) the reduction was major that in M. truncatula var. Jemalong (salt tolerance). Moreover, this effect was significantly more severe in plants subjected to mannitol. On the contrary, (Seckin et al., 2009) observed in salt sensitive wheat decreased growth under salt stress, but this effect was alleviated by mannitol pretreatment. Nitrogen fixation rate (NFR) showed a behavior similar to the described one for the PDW, in fact plants of M. truncatula treated with 100 mM NaCl showed inhibition by 48% in salt tolerance variety and by 66% in salt sensitive variety, however with 100 mM mannitol inhibition was by 40% in salt tolerance variety and by 98% in salt sensitive variety. The inhibition produced by mannitol has been previously reported (Mhadhbi et al., 2009) in M. truncatula. This sensitivity of nitrogenasa activity compared to plant growth was explained by the complexity of interaction and the need for energy required by the nodule. Regarding to enzyme activities of ammonium metabolism in nodules, glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in M. truncatula var. R108 (salt sensitive) were higher than M. truncatula var. Jemalong (salt tolerance) (Figure 2). Under salt and osmotic stress, GS and GOGAT activities decreased significantly in both varieties, except in nodules of R108 expose to 100 mM mannitol where enhanced by 55% (GS) and by 22% (GOGAT). In bibliography has been reported that the role of GDH under some environmental conditions cannot be dismissed (Srivastava and Singh, 1987). This enzyme may play an alternative role to the GS/GOGAT cycle in ammonium assimilation under specific physiological conditions that make the ammonium concentration increase (Robinson et al., 1991). In our study, under salt stress this activity increased in both varieties, this result showed clearly that GDH became the predominant ammonium assimilating way under salt stress in M. truncatula. This activity in R108 (salt sensitive) increased under mannitol stress too, but decreased in Jemalong (salt tolerance). Accumulation of cyclic polyols such as myoinositol or pinitol has frequently been reported in response to drought and salinity (Vernon and Bohnert, 1992; Streeter et al., 2001). In nodules of Jemalong, myoinositol and pinitol contents increased with salt stress, whereas in nodules of mannitol stressed plants showed a decrease compared to nonstressed plants. On the other hand, in nodules of R108 both polyols increased under salt and osmotic stress, nevertheless we observed the highest content with 100 mM mannitol, 4.5 times myoinositol and 11 times pinitol. As expected, mannitol content enhanced in nodules of both varieties under mannitol stress, although it was more important in R108 than Jemalong. According our data, we could suggest that glutamate dehydrogenase and polyols have a central function in the adaptative response of the nodule to salt and osmotic stress. REFERENCES Cataldi T, et al. Analytical Chemistry 2000;72:3902-3907. Groat RG, Vance CP. Plant Physiology 1981;67:1198-1203. Mhadhbi H, et al. J Agro and Crop Science 2009;195:225-231. Munns R, Tester M. Annual Review Plant Biology 2008;59:651-681. Robinson SA, et al. Plant Physiology 1991;95:509-516. Seckin B, et al. Journal of Plant Growth Regulation 2009;28:12-20. Singh RP, Srivastava HS. Physiologia Plantarum 1986;66:413-416. Srivastava HS, Singh RP. Phytochemistry 1987;26:597-610. Streeter JG, et al. Plant Cell and Environment 2001;24:429-438. Vernon DM, Bohnert HJ. Embo Journal 1992;11:2077-2085. Wallsgrove RM, et al. Plant Physiology 1979;63:232-236. Witty Jf, Minchin FR. Journal of Experimental Botany 1998;49:1041-1047. Zhu JK. Trends in Plant Science 2001;6:66-71 This work was financed by proyect AGL 2008-00155/AGR