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Contribution des phytoglobines et des nitrate réductases à la régulation de l’oxyde nitrique et de la fixation de l’azote dans la symbiose Medicago truncatula / Sinorhizobium meliloti

Abstract : The nitrogen-fixing symbiosis between legumes and soil bacteria of Rhizobia type reduces atmospheric nitrogen (N2) to ammonia (NH3) through the presence of bacterial nitrogenase in a root organ, called nodule.In the symbiotic model Medicago truncatula / Sinorhizobium meliloti, nitric oxide (NO) is produced throughout the symbiotic process, from the beginning of the interaction between the plant and the bacteria until the senescence of the nodule. The toxic, signal or metabolite effects of NO depend mainly on its concentration at the action site. Its concentration within the nodule cells must be regulated in order to limit its toxic effects and leads its signaling and metabolite functions. In plants, the main sources of NO identified are nitrate reductase (NR) and the mitochondrial electron transfer chain (ETC). In addition, phytoglobins (Phytogb) are known to be involved in the catabolism of NO. According to their sequences homology and affinity for oxygen, three classes of Phytogb have been described in legumes: non-symbiotic Phytogb (Phytogb1), legume-specific leghemoglobin (Lb) and truncated Phytogb (Pgb3).The main objectives of this PhD were, on the one hand, to characterize and study the role of NR and Phytogb in the NO regulation, during the symbiosis between M. truncatula and S. meliloti and, on the other hand, to analyse the role of NO in the development and in mature nodule during the symbiotic process.In M. truncatula, 3 genes code for NR and 17 for Phytogb. The phylogenetic study of M. truncatula Phytogb identified 12 Lb, 3 Phytogb1 and 2 Phytogb3. Analysis of NR and Phytogb gene expression, as well as measurement of total NR activity and NO production, allowed to monitor the level of NO during the N2-finxing symbiosis, and to determine the respective roles of the different NR and Phytogb in the NO regulation. During the symbiotic process, four peaks of NO production were observed, corresponding to four periods of the symbiotic process, during (1) the establishment of the interaction between the plant and the bacteria, (2) at the setup of the nodule organogenesis (3) inside the functioning and mature nodule and (4) at the onset of nodule senescence. During these different periods, the production of NO is particularly correlated with the expression of the NR1 and 2, one Phytogb1 (Phytogb1.1) and one Phytogb3 (Phytogb3.1) genes. The use of various inhibitors of NO synthesis pathways has shown that NO production depends mainly on NR activity and the mitochondrial electron transfer chain. The use of NO donors has shown that, during nodular development, NO induces the expression of Phytogb1 and several defence genes but represses Lb and Phytogb3 genes. Functional analysis of Phytogb1.1, during nodule establishment, functioning and senescence, was initiated via the production of M. truncatula plants overexpressing or silencing this gene. Overexpression and under-expression of this gene resulted, respectively, in a decrease and an increase in the NO level in the nodules, but in both cases resulted in a decrease in the number of nodules per plant. Analysis of the expression of several markers genes of the symbiotic interaction, defence responses, nitrogen metabolism and hypoxia, and analysis of nitrogen fixation in mature nodules highlight the particular role of Phytogb1.1 in the NO regulation during nodule development and during the senescence process.
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Antoine Berger. Contribution des phytoglobines et des nitrate réductases à la régulation de l’oxyde nitrique et de la fixation de l’azote dans la symbiose Medicago truncatula / Sinorhizobium meliloti. Biologie végétale. Université Côte d'Azur, 2019. Français. ⟨NNT : 2019AZUR4006⟩. ⟨tel-02468326⟩

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