Functional divergence and symbiotic significance of nitrate reductase isoforms in <i>Medicago truncatula</i>

Nitrate reductase (NR) is a key enzyme in nitrate assimilation, yet its function within nodules remains poorly understood. In Medicago truncatula, three NR genes, MtNR1, MtNR2, and MtNR3, exhibit distinct evolutionary origins and regulatory features. Phylogenetic analyses indicate that NR3-type genes, originated from a duplication of NR1 within Inverted Repeat-Lacking Clade (IRLC) legumes, have lost the conserved phosphorylation sites critical for post-translational regulation. To assess the functional significance of these isoforms, we characterized single and double nr mutants obtained through Tnt1 transposon insertion under nitrate nutrition and during symbiosis. MtNR1 is the primary contributor to total NR activity: with nr1 and nr2 mutants retaining around 10% and 30% of wild-type levels, respectively. The nr1/nr2 double mutant shows an almost complete loss of NR activity and fails to survive under nitrate supply, demonstrating the essential and non-redundant roles of both isoforms. Under symbiotic conditions, single mutants displayed normal nodulation, whereas nodule development was nearly abolished in the double mutant despite continued MtNR3 expression. In addition to its role in nitrogen assimilation, single nr mutants showed increased sensitivity to hypoxic stress and impaired recovery of nitrogen fixation, revealing a role for NR in nodule energy metabolism through the phytoglobin-NO respiration pathway. We propose that the combined loss of NR1 and NR2 disrupts NO cycling linked to mitochondrial electron transport, thereby compromising the energy balance required for symbiosis under microoxic conditions. This work provides a framework to investigate NR diversification in legumes and opens perspectives for improving nitrogen fixation under environmental constraints.