Nitrate reductase activity is required inMedicago truncatula-Sinorhizobium melilotinitrogen-fixing symbiosis

AbstractNitrate 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 ofMtNR1,MtNR2andMtNR3inM. truncatula, three singleTnt1retrotransposon-taggednrmutants and onenr1/nr2double mutant were analyzed on plants growing either on nitrate, or during the nodulation process. Overall, the absence of phenotypes observed inM. truncatulasingle mutants suggests a significant functional redundancy between the different NRs inM. truncatula. The most striking outcome of this work is the almost complete impairment of nodulation capacity observed in thenr1/nr2double mutant, demonstrating that NR activity is required for the functioning of the N2-fixing symbiosis.