The VvDREB2A-VvGolS3 regulatory module enhances cold tolerance in Vitis vinifera L

Galactinol synthase (GolS), a crucial enzyme in the synthesis pathway of raffinose family oligosaccharides (RFOs), plays a vital role in plants against abiotic stress. In previous experiments, VvGolS3 cloned from the grape variety 'Zuoyouhong' exhibited cold-inducible expression. However, its specific function and regulatory mechanism remained largely unknown. Additionally, our research has shown that the transcription factor VvDREB2A was involved in grape response to cold stress, yet its molecular interaction with VvGolS3 required further investigation. In this study, it was observed that the expression level of VvGolS3 was significantly higher in cold-resistant grape varieties compared to sensitive ones. Overexpression of VvGolS3 in transgenic Arabidopsis resulted in enhanced cold tolerance, as evidenced by reduced levels of reactive oxygen species (ROS) and increased RFOs content under cold stress conditions. Transient overexpression of VvGolS3 in grapevine leaves also conferred enhanced cold tolerance. Furthermore, overexpressing-VvDREB2A grape calli showed increased cold tolerance and higher levels of RFOs. Similarly, transient overexpression of VvDREB2A in grapevine leaves enhanced cold tolerance. Through yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter system, it was confirmed that VvDREB2A directly binds to the VvGolS3 promoter and promotes its expression. Collectively, these findings indicate that VvDREB2A directly targetes and regulates the expression of VvGolS3, thereby promoting the accumulation of RFOs and enhancing antioxidant enzymes activity, and ultimately improving cold stress tolerance. This study provides the first mechanistic insight into VvGolS3-mediated cold resistance and identifies a novel genetic module (VvDREB2A-VvGolS3) for molecular breeding of cold-tolerant grape cultivars.