Epigenetic Silencing of MsMYB1R1 Drives Anthocyanin Reduction in Developing Red-Fleshed Apple

Red-fleshed apples have attracted widespread attention for their rich anthocyanin content. However, some red-fleshed apples, such as "Xiahongrou" (Malus sieversii), exhibit a decline in red pigmentation and anthocyanin content as the fruit develops. The molecular mechanism underlying this flesh discoloration is poorly understood. In this study, through transcriptome analysis, we identified an R1-type MYB transcription factor, MsMYB1R1, whose expression exhibited a positive correlation with alterations in flesh anthocyanins during fruit development. Overexpression of MsMYB1R1 significantly enhanced anthocyanin accumulation and upregulated anthocyanin biosynthesis-related structural gene expression through transient expression assays in apple fruit and stable transformation experiments in calli. Conversely, it significantly inhibited the expression of MsMYBL2, a known negative regulator of anthocyanin synthesis. In binding experiments, we demonstrated that MsMYB1R1 binds to the promoter of MsMYBL2 and inhibits its transcription. Additionally, we observed that the C⁵ DNA methylation level of the MsMYB1R1 promoter exhibited a gradual increase with fruit development, resulting in a concomitant decrease in its transcriptional level. Together, our results indicate that MsMYB1R1 functions as a repressor upstream of MsMYBL2. Elevated methylation levels in the MsMYB1R1 promoter inhibit its transcription, leading to a reduction in the anthocyanin content in the flesh during fruit develops.