The FaDOF2-FaMYB4 transcriptional module regulates anthocyanin biosynthesis in strawberry fruit.

key messageFaMYB4,encoding a core transcriptional repressor of anthocyanin biosynthesis, is suppressed by the upstream regulator FaDOF2, forming a novel cascade module that regulates strawberry fruit pigmentation. Strawberry fruit coloration is a key quality trait that is primarily determined by anthocyanin accumulation. In this study, we identified FaMYB4 as a core R2R3-MYB transcriptional repressor of anthocyanin biosynthesis, based on the high-quality haploid genome of octoploid cultivated strawberry 'Yanli' and corresponding transcriptome data across multiple fruit developmental stages. The transcription level of FaMYB4 gradually decreases during fruit ripening. Furthermore, FaMYB4 directly binds to the promoters of key anthocyanin biosynthetic genes (FaPAL, FaC4H, and FaANS) and represses their transcription, as demonstrated by DNA affinity purification sequencing, a yeast one-hybrid assay, an electrophoretic mobility shift assay, and a dual-luciferase reporter system. In addition, FaDOF2, a DOF family transcription factor, directly suppresses FaMYB4 transcription by binding to its promoter. In conclusion, our results reveal a FaDOF2-FaMYB4 cascade module that regulates anthocyanin biosynthesis in the octoploid cultivated strawberry, thereby extending the known regulatory network of fruit pigmentation and providing potential molecular targets for breeding improved color traits.