A dual legume-rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses.

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.

The Nitrate Assimilatory Pathway in Sinorhizobium meliloti: Contribution to NO Production.

The interaction between rhizobia and their legume host plants culminates in the formation of specialized root organs called nodules in which differentiated endosymbiotic bacteria (bacteroids) fix atmospheric nitrogen to the benefit of the plant. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobium-legume symbiosis where it has been shown to play multifaceted roles. It is recognized that both bacterial and plant partners of the Sinorhizobium meliloti-Medicago truncatula symbiosis are involved in NO synthesis in nodules. S. meliloti can also produce NO from nitrate when living as free cells in the soil. S. meliloti does not possess any NO synthase gene in its genome. Instead, the denitrification pathway is often described as the main driver of NO production with nitrate as substrate. This pathway includes the periplasmic nitrate reductase (Nap) which reduces nitrate into nitrite, and the nitrite reductase (Nir) which reduces nitrite into NO. However, additional genes encoding putative nitrate and nitrite reductases (called narB and nirB, respectively) have been identified in the S. meliloti genome. Here we examined the conditions where these genes are expressed, investigated their involvement in nitrate assimilation and NO synthesis in culture and their potential role in planta. We found that narB and nirB are expressed under aerobic conditions in absence of ammonium in the medium and most likely belong to the nitrate assimilatory pathway. Even though these genes are clearly expressed in the fixation zone of legume root nodule, they do not play a crucial role in symbiosis. Our results support the hypothesis that in S. meliloti, denitrification remains the main enzymatic way to produce NO while the assimilatory pathway involving NarB and NirB participates indirectly to NO synthesis by cooperating with the denitrification pathway.