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The current world population together with the predictions of further growth suggest that it is necessary to increase crop yields worldwide. Legumes are the second most important food crop after cereals, and thanks to their ability to establish a symbiotic relationship with soil bacteria, the impact of the use of nitrogen fertilizers on the environment is reduced. This symbiosis gives rise to the process known as biological nitrogen fixation (BNF), which consists in the reduction of molecular nitrogen to ammonium, from which plants synthesize organic nitrogenous products essential for their nutrition. Unfortunately, BNF is a very sensitive process to biotic and abiotic stresses such as salinity, drought, or nutrient limitation, among others. The general aim of this work was to gain further insights in the regulation of BNF and the physiological and biochemical mechanisms that plant activate in response to abiotic stresses. In order to counteract the negative effects of osmotic stresses, plant and bacteria are able to synthesise osmoprotectant compounds to maintain cell viability, e.g. the amino acid proline. A real-time monitoring of proline utilisation in both plant and bacterial systems is a first key step towards understanding the multiple roles of this molecule under osmotic stress situations. Our results in chapter one showed that, in bacteroids, proline accumulation does not occur during the stress phase, but during recovery, once optimal plant growth conditions are re-established. In chapter two, a proteomic and metabolic study was performed to gain further insights about amino acid metabolism in pea nodules. In the classical model of nutrient exchange between symbionts, plant supplies energy in the form of dicarboxylates to the N2-fixing bacteroids in exchange for ammonium. However, this classic model was challenged upon the observation that mutations in the general ABC amino-acid transporters AapJQMP and BraDEFGC in Rhizobium leguminosarum resulted in N starvation symptoms in both pea and bean plants. The uptake of branched-chain amino acids (BCAAs) from the plant by the bacteroid was found to be essential for an effective BNF at least in R. leguminosarum species. Another experimental approach to further understand the role of amino acid metabolism in nodules is the application of compounds that inhibit the biosynthesis of BCAAs in plant cells such as group B herbicides. These approaches allowed us to verify how the blockage of BCAA transport between symbionts had a greater effect on nodule metabolism than the inhibition of BCAA biosynthesis. In fact, BCAA biosynthesis was also inhibited due to the aap/bra double mutation. In chapter two, we also evaluate the effect of water deficit on nodule proteome, since among the strategies that plants use in response to abiotic stresses there are several related to amino acid metabolism. This study highlights the relevance of low abundant amino acids, such as methionine, aromatic amino acids or γ-aminobutyric acid, in the response to water deficit. Finally, until now no attempt has been made to carry out an integral approach in which possible changes caused by drought in carbon (C) allocation, and in addition, the effect on the consumption or accumulation of metabolites in all plant organs be analysed. For this purpose, in chapter three, the effect of drought on both the [U-13C]-sucrose distribution and ureides, organic acids and carbohydrates content were analysed. We found that drought decreased 13C transport to sink tissues and changed the priority of C allocation between sink organs. |
