An isoflavone reductase‐like protein <scp>MtIFL</scp> negatively regulates nodule symbiosis in <i>Medicago truncatula</i>

Flavonoids are valuable metabolites produced by legumes, including Medicago truncatula, and play crucial roles in signaling communication during legume-rhizobium symbiosis. Isoflavone reductase proteins (IFRs) are involved in the biosynthesis of isoflavones and plant defense regulation. However, their role in symbiotic nitrogen fixation remains mostly elusive. Here, 13 putative IFR gene family members were identified, and an IFR-like gene named MtIFL was functionally characterized through expression patterns, phenotypic characterizations, flavonoid metabolome, and transcriptome analyses. Furthermore, the binding partner of MtIFL was investigated using Y1H, EMSA, and Dual-LUC assays. MtIFL was strongly induced in the process of nodule development and expressed in the meristem and infection zone of mature nodules. Knockout of MtIFL promoted nodulation, while overexpression of MtIFL induced premature senescence of nodules. Further investigations revealed that MtIFL negatively regulates nitrogen-fixing symbiosis by mediating isoflavone metabolism, and MtNIN regulates MtIFL expression by binding to the hNRE motif. Overall, our findings suggest that MtIFL negatively regulates nodule formation, thereby influencing the survival of rhizobia in nodule cells. MtNIN regulates MtIFL expression and may play a role in isoflavone metabolism. These results provide novel insights into the function of IFRs in symbiotic nodulation and the role of flavonoids in nodule development.