Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing D 1-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia , and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula prolinerelated endogenous genes M. truncatula D

Nodulated bean plants were exposed to mild salt stress or water deficit in such a way that the nodule's nitrogen-fixing activity was reduced to about 25-30% that of controls. Water-deprived plants showed a slight decrease in the weight of the aerial part, whereas the photosynthetic parameters were not significantly affected. In contrast, saltstressed plants displayed a reversible decrease in the quantum yield of photosystem II photochemistry. Five waterdeficit responsive cDNA clones encoding one lipid transfer protein, two late-embryogenesis abundant (LEA) proteins and two proline-rich proteins (PRPs) showed different organ-specific expression patterns depending on the kind of stress applied. PRPs and one LEA protein, PvLEA-18, exhibited the highest expression in nodules. Anti-PvLEA-18 antibodies were used to immunolocalize the protein in the nodule. PvLEA-18 was localized in the cytoplasm and nucleus of nodule cortex cells, and preferentially in cells of the vascular bundles, showing enhanced accumulation under water deficit. To our knowledge, this is the first time that a LEA protein has been identified in legume nodules.