Transcription factor ABFs involved in regulating anthocyanin accumulation in‘Benibalado’ grape

Anthocyanins play a pivotal role in defining grape coloration which contribute to health-promoting properties of grape. Exogenous abscisic acid (ABA) plays a key role in promoting anthocyanin accumulation in grape berries. In this study, we investigated the molecular mechanism underlying ABA-regulated coloration in ‘Benibalado’ ( Vitis vinifera L.) grape skin through physiological, molecular, and phylogenetic analyses. After ABA treatment, the berry of grapes exhibited a significantly deeper red colour, accompanied by an increase of three additional anthocyanin types and a significant rise in the contents of Peonidin 3- O -glucoside and Malvidin 3- O -glucoside. ABA treatment significantly induced upregulation of ABA signaling genes ( VvABF1, VvABF2 and VvPYL1 ) and anthocyanin biosynthesis genes ( VvMYBA1, VvMYBA2, VvGST4 and VvGT1 ). A genome-wide analysis identified 64 bZIP transcription factors (TFs) in grape, which were classified into 13 subfamilies. Among these, VvABF1 and VvABF2 , belonging to subfamily A, exhibited distinct spatiotemporal expression patterns, with VvABF2 highly expressed in skin and flesh. Phylogenetic and motif analyses revealed evolutionary conservation and functional diversification among VvbZIPs. VvABF1 exhibits the closest phylogenetic relationship with LcABF1. VvABF2 showed the highest affinity with LcABF2 and LcbZIP32, followed by MdbZIP45 and MdbZIP90. Furthermore, subcellular localization confirmed the nuclear presence of both VvABF1 and VvABF2 . Yeast one-hybrid and dual-luciferase assays demonstrated that these TFs can active the activity of VvMYBA1 promoter. These results indicate that VvABFs mediate ABA-induced anthocyanin biosynthesis by directly regulating VvMYBA1 expression, providing new insights into the ABA- bZIP - MYBA regulatory module in grape berry coloration.