An activator-represssor complex of VvWRKYs regulate proanthocyanidins biosynthesis through co-targeting VvLAR in grape

Proanthocyanidins (PAs) are vital polyphenolic compounds in plants with various biological functions. Although WRKY transcription factors are known to play important roles, their specific involvement in regulating PAs metabolism in grapes remains underexplored. In this study, we identified six candidate WRKY genes potentially involved in PAs synthesis by transiently overexpressing them in Nicotiana tabacum leaves. Among these, VvWRKY57 was found to enhance PAs synthesis. Further functional analysis, achieved by overexpressing of VvWRKY57 in grape calli, confirmed its positive role in PAs biosynthesis. Using yeast one-hybrid (Y1H), dual-luciferase reporter (DLR) assays, and electrophoretic mobility shift assay (EMSA), we demonstrated that VvWRKY57 binds to the promoter of leucocyanidin reductase (VvLAR2) and stimulates its activity. Additionally, yeast two-hybrid (Y2H), bimolecular fluorescence complementary (BiFC), and pull-down assays revealed that VvWRKY57 forms heterodimers with VvWRKY20, while VvWTKY20 also forms homodimers. Interestingly, overexpression of VvWRKY20 was found to inhibit PAs synthesis. Y1H, DLR, and EMSA further showed that VvWRKY20 binds to the promoters of VvLAR1 and VvLAR2, repressing their transcription activity. When VvWRKY57 and VvWRKY20 were co-expressed, VvLAR2 promoter activity and PAs synthesis were suppressed. Moreover, we discovered that VvPUB26, an E3 ubiquitin ligase physically interacts with both VvWRKY57 and VvWRKY20. VvPUB26 mediated the degradation of VvWRKY20 but did not influence the degradation of VvWRKY57. In conclusion, this study highlights the regulatory interplay between WRKY transcription factors in PAs biosynthesis, offering insights into their distinct roles in modulating this important metabolic pathway in grapes.