Differences in nitrogen fixation between Vicia villosa Roth. and Vicia sativa

Vicia villosa Roth. (MM) and Vicia sativa (GG) are highly valued legume forages for their favorable nitrogen fixation, so they are widely used for soil fertility and improvement. Despite their benefits, no work has addressed the mechanism that underlies the differences in their nitrogen fixation. To fill this gap, the present study employed physiological, cytological, and comparative transcriptomic approaches to elucidate the disparities in nitrogen fixation between MM and GG. Our results showed that the total amount of nitrogen-fixed and the nitrogen response comprehensive performance by MM is significantly higher than GG, but nitrogen fixation efficiency is the same. A larger infection zone and a higher proportion of bacteroid differentiation were observed in mature nodules of MM. MM had a 0.201 pg larger genome than GG, in line with its larger biomass. Both species exhibited strong positive selection in their symbiotic nodulation processes, with nodule-specific cysteine-rich peptides (NCRs) constituting 88.9% of the identified genes. Significant enrichment of the flavonoid biosynthetic pathway was found only in MM-specific genes, among which chalcone-flavonone isomerase, caffeoyl-CoA-O-methyltransferase and stilbene synthase were extremely highly expressed. MM-specific genes also exhibited significant enrichment of symbiotic nodulation processes; genes relative to NCRs made up 61.1% of highly expressed genes and 62.5% of extremely highly expressed genes. qRT-PCR demonstrated that the higher enrichment and expression of the dominant NCR (Unigene004451) promoted higher bacteroid differentiation of nodules and higher nitrogen fixation in MM. Our findings suggest that the higher total nitrogen fixation of MM is attributed to MM's larger biomass, leading to higher nitrogen demand and enhanced fixation physiology. On a molecular level, this is explained by the synergistic effects of high bacteroid differentiation and high expression of NCRs.