Legumes such as soybeans, peas, and alfalfa form symbiotic relationships with nitrogen-fixing bacteria called rhizobia, which reside inside root nodules and convert atmospheric nitrogen into ammonia that plants can use. This natural fertilization process enriches soil and cuts the need for chemical fertilizers, making it a cornerstone of sustainable agriculture. Yet the molecular choreography behind nodule formation remains incompletely understood. Plant hormones, particularly auxin, are known to influence nodule initiation, but how the circadian clock—the master regulator that aligns biological processes with day-night cycles—fits into this picture has been a persistent puzzle. Given these challenges, there is a pressing need for in-depth research into how circadian regulation integrates with hormonal pathways to control nodulation and nitrogen fixation in legume crops.
Now, a team led by researchers at South China Agricultural University has uncovered a key piece of this puzzle. In a study published (DOI: 10.1093/hr/uhag124) April 6, 2026, in Horticulture Research, Tao Fan and colleagues demonstrate that the core clock gene MtLUX directly represses a downstream gene called MtRVE1, which in turn controls auxin biosynthesis. This regulatory cascade shapes both root architecture and nodule formation in the model legume Medicago truncatula, revealing a previously unknown mechanism by which the circadian clock governs symbiotic nitrogen fixation.
The researchers found that when MtLUX was overexpressed, plants produced significantly more nodules and showed enhanced nitrogenase activity—the enzymatic measure of nitrogen fixation—compared to wild-type plants. Conversely, mtlux mutants had fewer nodules and reduced nitrogenase activity. Using chromatin immunoprecipitation and electrophoretic mobility shift assays, the team confirmed that the MtLUX protein binds directly to specific sites on the MtRVE1 promoter, repressing its expression. MtRVE1 itself is a clock-output gene previously linked to auxin regulation in Arabidopsis. When the researchers knocked out MtRVE1, nodule numbers increased—the opposite of what happened when MtLUX was lost. Double mutants lacking both MtLUX and MtRVE1 phenocopied the mtrve1 single mutant, firmly placing MtRVE1 downstream of MtLUX in this regulatory pathway. Further analysis revealed that MtLUX modulates auxin levels through this cascade: mtlux mutants had reduced free auxin (indole-3-acetic acid) and auxin conjugates in their nodules, while mtrve1 mutants showed elevated auxin levels. The study also identified MtYUC5 and MtYUC10—key auxin biosynthesis genes—as downstream targets whose expression is suppressed when MtRVE1 is active.
“We were surprised to find that a core clock component directly controls nodule formation through auxin—it’s a connection that hadn’t been made before in legumes,” the authors said. “The circadian clock has long been known to regulate above-ground traits like flowering and growth, but its role below ground, particularly in the context of nitrogen-fixing symbiosis, was largely a black box. Our work shows that the MtLUX-MtRVE1 module acts as a molecular bridge, translating daily timekeeping signals into hormonal cues that shape root development. This opens up a whole new way of thinking about how plants coordinate their internal rhythms with their nutritional needs.”
The findings have practical implications for agriculture and crop breeding. By understanding how the circadian clock influences nodulation, scientists could potentially develop legume varieties with enhanced nitrogen-fixing capacity, reducing the need for nitrogen fertilizers—a major source of greenhouse gas emissions and water pollution. The study also suggests that planting time and light exposure could influence nodulation efficiency, offering an immediate, low-cost strategy for optimizing symbiotic nitrogen fixation in the field. More broadly, the MtLUX-MtRVE1-MtYUC pathway provides a new target for genetic improvement of legumes, from soybeans to alfalfa, helping to secure food production in a changing climate.
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References
DOI
10.1093/hr/uhag124
Original Source URL
https://doi.org/10.1093/hr/uhag124
Funding information
This work was supported by the National Key R&D Program of China (2024YFD1200803), Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops (FCBRCE-202518), National Natural Science Foundation of China (32570281 and 32400226), Natural Science Foundation of Guangdong Province (2024A1515011112, 2026A1515010765 and 2026A1515010679), Guangdong Basic and Applied Research (2023A1515110413), China Postdoctoral Science Foundation (2024M750953).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.