The VvWRKY26-MBW Complex Induced by Salicylic Acid Promotes the Accumulation of Proanthocyanidins in Grape

Proanthocyanidins (PAs) are a significant class of polyphenolic compounds found in grapes, playing important roles in human health and plant stress resistance. Previous studies have shown that the VvMYBPA1/PA2-VvWDR1-VvMYC2 (MBW) complex can regulate the biosynthesis of proanthocyanidins, and some studies have shown that the homologous genes of VvWRKY26 are involved in the biosynthesis of proanthocyanins and anthocyanins in Arabidopsis thaliana and petunias, but the molecular mechanism of VvWRKY26 in regulating the biosynthesis of proanthocyanins in grapes is still unclear. In this study, we found that the content of proanthocyanidins and the expression of related structural genes were significantly increased by salicylic acid (SA) incubation in grapes during the color transition period. Overexpression of VvWRKY26 in grapevine healing tissues revealed that overexpression of VvWRKY26 significantly promoted the accumulation of proanthocyanidins and up-regulation of related structural genes when compared with the empty vector. Further investigation into the interaction mechanisms through yeast two-hybrid and bimolecular fluorescence complementation assays revealed that VvWRKY26 can interact with VvMYBPA1/PA2, VvMYC2, and VvWDR1 to form VvMYBPA1/PA2-VvWDR1-VvMYC2-VvWRKY26 (W-MBW) complex. Through yeast one-hybrid assays and dual-luciferase reporter analysis, it was confirmed that VvWRKY26 could bind to the promoters of VvANR and VvLAR2 and activate their activity. Finally, through the co-overexpression of VvWRKY26 and MBW complex, it was discovered that the promoting activity of VvANR and VvLAR2, as well as the biosynthesis of PAs, were significantly enhanced, which was much higher than the effect of the MBW complex alone, while the opposite occurred after co-interference. In conclusion, this study explored the role of VvWRKY26 in the biosynthesis of proanthocyanidins in grapes after the interaction with the MBW complex to form W-MBW under SA incubation, providing a new regulatory mechanism for the biosynthesis of proanthocyanidins in grapes.