DaMYB75 and DaMYB56 antagonistically regulate anthocyanin biosynthesis by binding to the DaANS promoter in Dioscorea alata

The yam Dioscorea alata L. is widely cultivated globally. Purple-fleshed varieties of this important crop have enhanced market value due to their high anthocyanin contents, but how anthocyanin biosynthesis in D. alata tubers is regulated remains poorly understood. In this study, we identified and functionally validated key transcription factors that regulate anthocyanin biosynthesis based on a comparative transcriptome and metabolome analysis of three D. alata cultivars with different colored tubers (dark purple, light purple, and white). The anthocyanin glycoside cyanidin-3-O-(2′′-O-glucosyl) glucoside was abundant during early tuber development, and we determined that its accumulation is regulated in opposite manners by two R2R3-MYB transcription factors: DaMYB75 and DaMYB56. Yeast two-hybrid and bimolecular fluorescence complementation assays in Nicotiana benthamiana and co-expression assays in D. alata demonstrated that DaMYB75 promotes anthocyanin biosynthesis by specifically activating the promoter of the late anthocyanin biosynthesis gene DaANS and enhancing its expression through an interaction with DabHLH72. By contrast, DaMYB56 is a negative regulator of anthocyanin biosynthesis that binds to the DaANS promoter together with DabHLH72. Furthermore, the methylation levels of the DaMYB75 promoter were significantly lower in purple tubers than in white tubers. These findings shed light on the regulation of anthocyanin biosynthesis by MYBs and provide the basis for genetically improving anthocyanin content in D. alata.