The VvPUB8–VvbHLH93–VvMYB15/VvMYB5a module inhibits the synthesis of anthocyanins in grape in response to MeJA

Anthocyanins are a critical component influencing the quality of grape. At varying concentrations, methyl jasmonate (MeJA) shows a concentration-dependent effect on the anthocyanin content in grapes. However, its molecular mechanism is unclear. In this study, we characterized an E3 ubiquitin ligase VvPUB8 that responds to MeJA and verified its negative regulation of grape anthocyanin synthesis through overexpression and mutant vectors' transformation of "Gamay" calli. Furthermore, VvPUB8 interacted directly with the transcription factor VvbHLH93, which can positively regulated anthocyanin synthesis by activating the promoters of VvMYB15 and VvMYB5a. The stability or activity of proteins regulated by ubiquitination largely depends on the type and number of the attached ubiquitin. Here, we showed that VvPUB8 facilitated K6- and K33-linked ubiquitination of VvbHLH93, thereby promoting VvbHLH93 degradation. Exogenous MeJA accelerated VvbHLH93 protein degradation and inhibited VvMYB15 promoter activation. Consequently, the synthesis of grape anthocyanins was suppressed. This study revealed that in response MeJA, VvPUB8 regulates VvbHLH93 stability through conjugation of distinct polyubiquitin chains, thereby modulating VvMYB15 and VvMYB5a promoter activity, thus inhibiting anthocyanin synthesis.