Protein phosphatase 2Cs (PP2Cs) are members of the serine/threonine phosphatase family that play pivotal roles in regulating plant development and responses to environmental stresses. However, comprehensive genome-wide studies of the PP2C gene family in grape (Vitis vinifera L.) have not yet been conducted. In the present study, 78 VvPP2C genes were identified and classified into 12 clades based on their phylogenetic relationships. Analysis of physicochemical properties and gene/protein architectures revealed that the members within each clade shared conserved structural features. Synteny analysis demonstrated that both tandem and segmental duplications substantially contributed to the expansion of the VvPP2C gene family. Tissue-specific transcriptional profiles and cis-element analyses indicated the potential involvement of these genes in grape development and stress responses. Moreover, expression analysis identified VvPP2C26 and VvPP2C41 as the most abscisic acid (ABA)-responsive genes, with expression patterns highly correlated with grape berry development. Functional validation in transgenic tomato lines demonstrated that the overexpression of either gene markedly delayed fruit ripening. Collectively, this study provides new insights into the evolutionary diversification and regulatory functions of the PP2C gene family in grape and identifies VvPP2C26 and VvPP2C41 as key candidates for elucidating ABA-mediated ripening mechanisms in non-climacteric fruits.

Soil salinity severely limits grapevine yield and fruit quality; however, the mechanisms underlying salt tolerance remain poorly understood. Small secreted peptides, particularly rapid alkalinization factor (RALF) peptides, are key regulators of plant responses to environmental stress. However, a systematic analysis of the RALF gene family in grapevine (Vitis vinifera L.) has not been conducted. In this study, we identified 11 VvRALFs in the grapevine genome (PN_T2T) and characterized their physicochemical properties, structural features, and evolutionary relationships. Expression profiling revealed that VvRALF9 was consistently upregulated in the roots of the salt-sensitive rootstock ‘Beta’ but downregulated in the salt-tolerant rootstock ‘3309C’. Silencing VvRALF9 in the roots of grapevine rootstocks significantly enhanced their salt tolerance. Protein interaction assays, including yeast two-hybrid, split-LUC, and pull-down assays, identified VvFER2 as a potential receptor of VvRALF9. Overexpression of VvFER2 in the roots of grapevine rootstocks also improved salt tolerance. Transcriptomic analysis of VvRALF9-RNAi roots revealed significant enrichment of the plant hormone signal transduction pathway, together with extensive changes in the expression of transcription factors and genes associated with multiple hormone signaling pathways. Together, these findings suggest that the VvRALF9–VvFER2 module is involved in the regulation of grapevine salt stress responses.