VvBBX44 and VvMYBA1 form a regulatory feedback loop to balance anthocyanin biosynthesis in grape.

Anthocyanins are essential for the quality of perennial horticultural crops, such as grapes. In grapes, ELONGATED HYPOCOTYL 5 (HY5) and MYBA1 are two critical transcription factors that regulate anthocyanin biosynthesis. Our previous work has shown that B-box protein 44 (VvBBX44) inhibits anthocyanin synthesis and represses expression in grape calli. However, the regulatory mechanism underlying this regulation was unclear. In this study, we found that loss of function resulted in increased anthocyanin accumulation in grapevine callus. VvBBX44 directly represses , which activates . VvMYBA1, but not VvBBX44, directly modulates the expression of grape UDP flavonoid 3--glucosyltransferase (). We demonstrated that VvBBX44 represses the transcriptional activation of and induced by VvMYBA1. However, VvBBX44 and VvMYBA1 did not physically interact in yeast. The application of exogenous anthocyanin stimulated expression in grapevine suspension cells and tobacco leaves. These findings suggest that VvBBX44 and VvMYBA1 form a transcriptional feedback loop to prevent overaccumulation of anthocyanin and reduce metabolic costs. Our work sheds light on the complex regulatory network that controls anthocyanin biosynthesis in grapevine.