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.

The postharvest senescence phase of table grapes comprises a series of biological processes. MicroRNAs (miRNAs) regulate downstream genes at the post-transcriptional level; however, whether miRNAs are involved in postharvest grape senescence remains unclear. We used small RNA sequencing to identify postharvest-related miRNAs in ‘Red Globe’ (Vitis vinifera) grapes harvested after 0, 30, and 60 d of storage at 4°C (RG0, RG30, RG60). In total, 42 known and 219 novel miRNA candidates were obtained. During fruit senescence, the expression of PC-3p-3343_1921, miR2950, miR395k, miR2111, miR159c, miR169q, PC-5p-1112_4500, and miR167b changed significantly (P<0.05). Degradation sequencing identified 218 targets associated with cell wall organization, tricarboxylic acid (TCA) cycling, pathogen defense, carbon metabolism, hormone signaling, the anthocyanin metabolism pathway, and energy regulation, of which ARF6, GRF3, TCP2, CP1, MYBA2, and WRKY72 were closely related to fruit senescence. We also verified that VIT_00s2146g00010, VIT_02s0012g01750, and VIT_03s0038g00160 with unknown functions are cleaved by senescence-related PC-5p-1112_4500 via the dual luciferase assay, and the transient transformation of grape berries showed that they regulate berry senescence. These results deepen our understanding of the role of miRNAs in regulating grape berry senescence and prolonging the shelf life of horticultural products. Based on these results, we propose a new theoretical strategy for delaying the postharvest senescence of horticultural products by regulating the expression of key miRNAs (e.g., PC-5p-1112_4500), thereby extending their shelf life.