Molecular evolution of RAXs in Vitis vinifera and functional analysis of VvRAX82 gene under salt stress

MYB transcription factors play pivotal roles in regulating plant growth and development, as well as enhancing plant resistance to both biotic and abiotic stresses. Among them, RAXs (Regulators of Axillary Meristems), a subgroup of the R2R3-MYB family, exhibit active responsiveness to abiotic stresses in plants. However, the phylogenetic relationships within the RAX gene family and the molecular mechanisms underlying salt stress response mediated by VvRAX82 in grapevine have not yet been fully elucidated. In this study, we identified 93 RAX genes in grapevine, distributed across all 20 chromosomes and classified into four distinct subfamilies. Promoter analysis revealed abundant stress-responsive cis-regulatory elements in their regulatory regions. qRT-PCR analysis revealed that VvRAX82 expression was significantly induced by salt stress. Subcellular localization demonstrated nuclear targeting of VvRAX82, and yeast trans-activation assays confirmed its transcriptional activation activity. Transient overexpression of VvRAX82 in grapevine leaves significantly reduced H₂O₂ and MDA accumulation, enhanced Pro content, photosynthetic parameters, and mitigated stress-induced cellular damage. The overexpression simultaneously induced the upregulation of major salt-stress-responsive genes, specifically the VvSOS1, VvSOS2, VvSOS3 and VvAHA2. Overexpression of VvRAX82 in A.thaliana and grapevine calli significantly reduced MDA and H₂O₂ accumulation while elevating the activities of key antioxidant enzymes (SOD, POD, and CAT) and upregulating salt-tolerance genes (e.g., AtKAT1, AtAPX1). Transcriptome profiling further revealed that VvRAX82 overexpression upregulated pathway-enriched genes in plant hormone signal transduction (49 DEGs) and carbon metabolism (35 DEGs). This study represents the first systematic analysis of evolutionary relationships within the grape RAX gene family and demonstrates that VvRAX82 enhances salt tolerance by strengthening antioxidant defenses, modulating salt signaling pathways, and activating carbon metabolism. Collectively, these findings establish a theoretical foundation for RAX gene functional studies and provide candidate targets for molecular breeding of salt-tolerant crops.