Nitrate reductase activity is required in Medicago truncatula-Sinorhizobium meliloti nitrogen-fixing symbiosis

Nitrate reductase (NR) is a key enzyme in higher land plants, catalyzing the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Phylogenetic analysis of NR protein sequences indicates that duplication events responsible for the existence of two NR branches, corresponding to NR1 and NR2 genes, occurred after the divergence of the different orders within the Rosids clade. A third NR sequence branch, named NR3-type, emerged in the inverted repeat-lacking clade of the Fabales order. An intriguing feature of the NR3-type sequences is the absence of conserved phosphorylation sites in the two hinge regions, in contrast to all other NRs. To investigate the respective roles of MtNR1, MtNR2 and MtNR3 in M. truncatula, three single Tnt1 retrotransposon-tagged nr mutants and one nr1/nr2 double mutant were analyzed on plants growing either on nitrate, or during the nodulation process. Overall, the absence of phenotypes observed in M. truncatula single mutants suggests a significant functional redundancy between the different NRs in M. truncatula. The most striking outcome of this work is the almost complete impairment of nodulation capacity observed in the nr1/nr2 double mutant, demonstrating that NR activity is required for the functioning of the N2-fixing symbiosis.