A novel mechanism of VvMYB308 in regulating anthocyanin biosynthesis in the leaves of Red-Fleshed grapevine genotypes

Grape (Vitis L.) is widely regarded as one of the most economically significant fruit trees on a global scale. The color of the fruit directly impacts its commercial value. Among the currently cultivated grape varieties, the flesh is predominantly white, with some varieties exhibiting red flesh. The majority of Red-Fleshed grapes undergo a reddening of their leaves in the autumn; however, there is a paucity of studies investigating the molecular mechanism of reddening of grape leaves. This study revealed a novel mechanism of VvMYB308 in the anthocyanin synthesis pathway of grapes: as evidenced by multiple sequence comparisons which showed the highest degree of homology with CcMYB308 in Citrus clementina. The results of subcellular localization showed that VvMYB308 protein was localized in the nucleus; the results of Y1H and DLR assays verified that VvMYB308 could bind to the VvCHI promoter and effectively activate its transcriptional activity; while the Y2H, BiFC, and GST Pull-down assays revealed that there was an interaction between VvMYBA6 and VvMYB308.DLR assay after co-expression of VvMYBA6 and VvMYB308 revealed that VvMYBA6 significantly activated the transcriptional activity of VvMYB308 on the VvCHI promoter, while VvMYB308 repressed the transcriptional activity of VvMYBA6 on the VvUFGT promoter. This finding indicates that VvMYB308 may act as a negative feedback regulator during anthocyanin metabolism. In summary, the present study revealed that VvMYB308 is involved in the regulation of anthocyanin synthesis in Red-Fleshed grape leaves by activating the transcriptional activity of VvCHI. Furthermore, evidence was provided that VvMYB308 formed a dynamic regulatory network with VvMYBA6 which synergistically activates the expression of VvCHI and represses the transcriptional activity of VvUFGT. This study thus reveals the fine balance of anthocyanin metabolism in Red-Fleshed grapes. The study has elucidated the precise equilibrium mechanism of anthocyanin metabolic processes, thus establishing a pivotal objective for the molecular breeding of Red-Fleshed cultivars.