Objectives The symbiotic nodule system established by leguminous plants and rhizobia stands as one of the most exemplary mutualistic symbioses in nature, constituting the central mechanism underlying efficient biological nitrogen utilization in legume crops. Fulvic acid, a naturally occurring organic small molecule with bioactivity, has been demonstrated to significantly enhance nodule formation, nitrogen fixation efficiency, and crop yield in leguminous plants, however, its specific mechanism of action remains unclear. This study investigates the mechanism by which FA promotes nodulation and nitrogen fixation in soybean from the perspective of nitrogen metabolism.

Methods A pot experiment was conducted using double-layer cups. The upper plastic cup was filled with vermiculite as the growth substrate, and the lower glass cup contained a low-nitrogen nutrient solution. After transplanting soybean seedlings, rhizobia were inoculated into the substrate, and the nutrient solution was supplemented with 0, 300, 500, or 1000 mg/L of FA, corresponding to the CK, H300, H500, and H1000 treatments, respectively. At different growth stages, seedling growth, chlorophyll content, nodule number, and leghemoglobin content in the nodules were measured. Nodule samples from the CK and H500 treatment groups collected on day 16 and day 25 were subjected to transcriptome sequencing. Clean reads obtained after quality control were mapped to the soybean genome, followed by functional annotation and enrichment analysis using GO and KEGG to identify differentially expressed genes (DEGs) associated with nitrogen metabolism pathways. Based on the 500 mg/L FA treatment (HA), an additional treatment with the NO inhibitor cPTIO was set up. Related seedling and nodulation indicators were measured, and the key products and enzyme activities in the root nitrogen metabolism pathway were analyzed.

Results Compared with CK, the H500 treatment increased nodule number by 73.12% and 46.51%, and leghemoglobin content in nodules by 45.38% and 25.50% at 16 days and 25 days, respectively. Chlorophyll content increased by 45.36%, 27.86%, and 12.64% at 5 days, 9 days, and 14 days, respectively. The H300 treatment showed no significant difference from CK, while H1000 inhibited seedling growth. Compared with CK, 428 and 2406 DEGs were identified in the H500 nodules at 16 days and 25 days, respectively, among which 15 and 27 DEGs were annotated to nitrogen metabolism pathways. Compared to HA, the HA+cPTIO treatment significantly inhibited seedling growth, root nodulation, and chlorophyll content; decreased root NO, nitrate nitrogen, and free amino acid contents; but increased nitrite content. Compared to CK, FA stimulated nitrate reductase (NR) activity, inhibited glutamate dehydrogenase (GDH) activity and free amino acid content, accelerated NO₃⁻-N reduction, and decreased nitrate and nitrite nitrogen contents.

Conclusions An appropriate concentration of fulvic acid can effectively up-regulate the expression of nitrogen metabolism-related genes in soybean root nodules, such as those encoding leghemoglobin, nitrate reductase, and asparagine synthetase (AS), while down-regulating the expression of the GDH gene. This subsequently regulates the glutamine synthetase/glutamate synthetase (GS/GOGAT) cycle system, increases the content of leghemoglobin and the activity of nitrate reductase, accelerates the scavenging of nitric oxide (NO), reduces the content of NO₃⁻-N, enhances the transport capacity of amino acids, and thereby improves plant growth and nodulation ability, as well as the nitrogen fixation efficiency of root nodules.