Analysis of R2R3-MYB repressor subfamily identifies FcMYB57 as a negative regulator of anthocyanin biosynthesis in Ficus carica L. fruit

Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries. Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig production. R2R3-MYB transcription factors have promotive or repressive regulatory roles in plant anthocyanin biosynthesis. In this study, 113 R2R3-MYB genes were identified in Ficus carica, 3 of which were assigned to the S4 subfamily of flavonoid-biosynthesis repressors. FcMYB57 was further recruited as a candidate anthocyanin-biosynthesis repressor based on its sequence features and expression, which was significantly negatively correlated with that of anthocyanin-biosynthesis structural genes. Transient overexpression of FcMYB57 in strawberry totally inhibited fruit pigmentation and significantly increased fruit firmness. The metabolomic analysis confirmed a significant reduction in the contents of cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside, as well as other flavonoids, and transmission electron microscopy revealed an increment in cell-wall thickness. Transcriptome analysis showed downregulation of anthocyanin-biosynthesis structural genes and upregulation of genes related to xylan synthesis. Yeast one-hybrid and dual luciferase assays demonstrated a negative regulatory effect of FcMYB57 on the promoter of FcUFGT3 (UDP glucose-flavonoid 3-O-glcosyl-transferase). Yeast two-hybrid assay showed that FcMYB57 does not interact with FcbHLH42, 3, 14, MYC2, or FcTTG1, all of which have a previously identified or predicted role in flavonoid biosynthesis, however, interaction was detected with FcTPL (Topless), suggesting that FcMYB57 serves as an active repressor of anthocyanin biosynthesis. This is the first identification of an anthocyanin-biosynthesis repressor in fig, with a possible role in fig fruit quality.