Transcriptomic insights into the potential impacts of flavonoids and nodule-specific cysteine-rich peptides on nitrogen fixation in Vicia villosa and Vicia sativa.

Vicia villosa (VV) and Vicia sativa (VS) are legume forages highly valued for their excellent nitrogen fixation. However, no research has addressed the mechanisms underlying their differences in nitrogen fixation. This study employed physiological, cytological, and comparative transcriptomic approaches to elucidate the disparities in nitrogen fixation between them. Our results showed that the total amount of nitrogen fixed was 60.45% greater in VV than in VS, and the comprehensive nitrogen response performance was 94.19% greater, while the nitrogen fixation efficiency was the same. The infection zone and differentiated bacteroid proportion in mature VV root nodules were 33.76% and 19.35% greater, respectively, than those in VS. The size of the VV genome was 15.16% larger than that of the VS genome, consistent with its greater biomass. A significant enrichment of the flavonoid biosynthetic pathway was found only for VV-specific genes, among which chalcone-flavonone isomerase, caffeoyl-CoA-O-methyltransferase and stilbene synthase were extremely highly expressed. The VV-specific genes also exhibited significant enrichment in symbiotic nodulation; genes related to nodule-specific cysteine-rich peptides (NCRs) comprised 61.11% of the highly expressed genes. qRT‒PCR demonstrated that greater enrichment and expression of the dominant NCR (Unigene0004451) were associated with greater nodule bacteroid differentiation and greater nitrogen fixation in VV. Our findings suggest that the greater total nitrogen fixation of VV was attributed to its larger biomass, leading to a greater nitrogen demand and enhanced fixation physiology. This process is likely achieved by the synergistic effects of high bacteroid differentiation along with high expression of flavonoid and NCR genes.