Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33

Anthocyanin is one of the most important pigments and nutrients in fruits. Genes encoding R2R3-MYB transcription factors (TFs) are key to anthocyanin regulation. R2R3-MYB activators have been widely studied in the anthocyanin synthesis pathway. However, the mechanism by which R2R3-MYB repressors negatively regulate anthocyanin synthesis remains poorly understood. In the present study, we characterized a subfamily 1 R2R3-MYB anthocyanin repressor gene, MYB306-like, from apple (Malus x domestica) and demonstrated its significance in anthocyanin regulation. The MdMYB306-like protein activates the expression of an anthocyanin repressor gene, MdMYB17, and inhibits the expression of MdDFR (an anthocyanin structural gene) by directly binding to the corresponding promoters. The MdMYB306-like protein interacts with MdMYB17 and MdbHLH33 through its N-terminus. In addition, MdMYB306-like interacts with MdbHLH33 and MdMYB17 to enhance its regulatory activities on MdMYB17 and MdDFR, respectively. Our results revealed that the MdMYB306-like protein, in conjunction with MdMYB17 and MdbHLH33, forms a key regulatory module to fine-tune anthocyanin synthesis in apple.