The VvMYBA1-Activated Electron Carrier VvCB5D Directs Flavonoid Metabolic Flux toward the Trihydroxylated Branch in Grape

Abstract Flavonoid β-ring hydroxylation influences fruit coloration, stability, and antioxidant capacity. Cytochrome P450 hydroxylases require electron-transfer partners, but their coordination during ripening remains unclear. Integrated transcriptomic and targeted metabolomic analyses identified the endoplasmic reticulum-localized cytochrome b5 VvCB5D as a VvMYBA1 co-expressed gene in grape (Vitis vinifera). VvMYBA1 directly activated VvCB5D transcription by binding its promoter, and VvCB5D interacted with VvF3′H and VvF3′5′H, preferentially with VvF3′5′H. Genetic manipulation in grape berries, tobacco leaves, and transgenic tomatoes showed that VvCB5D directed flavonoid flux toward the 3′,4′,5′-trihydroxylated branch, increasing delphinidin, petunidin, and malvidin derivatives. These results support a model in which VvMYBA1 coordinates flavonoid-pathway gene expression with a cytochrome b5 partner and identify VvCB5D as a candidate for modifying grape flavonoid composition.