Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

A challenging question is to define more precisely when and where reactive species are generated and to develop adapted tools to detect their production in vivo. To investigate the role of Noxs and NRs in the production of H(2)O(2) and NO, respectively, the use of mutants under the control of organ-specific promoters will be of crucial interest. The balance between ROS and NO production appears to be a key point to understand the redox regulation of symbiosis.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure.Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia.Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process.Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction.