Genome-wide analyses of RWP–RK reveal a potential role for a key gene, VvNLP1.1, in the grapevine response to nitrate

Nitrate is the primary nitrogen source for plants and is a signaling molecule regulating various plant developmental processes. Despite its significance, limited information is available on nitrate signaling in Vitis vinifera. We identified nine VvRWP–RK genes distributed across eight chromosomes using genome–wide identification and evolutionary analyses. Among these, VvNLP1–4 and VvRKD1–5 are associated with nitrate signaling and reproductive growth, respectively. To investigate their potential functions, structures, cis–acting promoter elements, functional structural domains, phylogenetic trees, spatiotemporal expression levels in different tissues at different developmental stages, potential protein–protein interaction networks, synteny (gene content), collinearity (gene order), and three–dimensional protein structure prediction were explored. We found that long–term nitrate application dramatically promoted grapevine plantlet development, including primary root length and leaf growth, and VvNLP1.1, VvNLP1.2, and VvNLP2 were highly expressed in ‘Thompson Seedless' root tissues under nitrate–enriched conditions. To clarify the critical role of nitrate in grapevine growth, we observed that nuclear localization of VvNLP1.1 increased significantly following nitrate treatment. VvNLP1.1 was found to bind to the promoter of the primary nitrate response gene VvNRT1.1, driving its transcriptional activity. These findings indicate that VvNLP1.1 is a core transcription factor of the nitrate signaling pathway in grapevine. Nitrate molecular docking analysis revealed that VvNLP1.1 directly binds to nitrate ions, indicating its potential role as a nitrate sensor capable of directly perceiving nitrate concentration. We also discovered that short–term nitrate starvation impacts VvNLP1.1 promoter activity, linked to the abscisic acid–binding element (ABRE) motif in its promoter region. Our results thus provide new insights into the molecular mechanisms underlying various physiological processes in grapevine, particularly the nitrate signaling pathway, and provide a theoretical basis for improving nitrogen use efficiency (NUE) in grapevine.