The circadian clock synchronizes a multitude of biological events with environmental changes, thereby optimizing plant growth and development. In legumes, nodule formation, a pivotal process that sustains symbiotic nitrogen fixation, is one such event regulated by the circadian clock. Nevertheless, the mechanisms underlying the circadian clock’s regulation of nodule formation and nitrogen fixation are still poorly elucidated. Herein, we unveil that the core clock gene LUX ARRHYTHMO (LUX) exerts a crucial role in modulating nodule formation and root development via auxin biosynthesis pathways in the model legume Medicago truncatula. Our findings indicate that MtLUX directly associates with the promoter of MtRVE1, a clock output gene involved in auxin biosynthesis, both in vivo and in vitro, thereby repressing its expression. Biochemical and genetic data further corroborate that the MtLUX-MtRVE1 regulatory module adjusts root architecture and nodule formation through the fine-tuning of auxin biosynthesis. These discoveries reveal a mechanism whereby the circadian clock integrates hormonal pathways to regulate nodule formation, thereby linking circadian regulation, auxin biosynthesis, and nitrogen fixation in legumes. This research lays the groundwork for enhancing legume growth and nitrogen acquisition under fluctuating environmental conditions.

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