Mdm-miR858 targets MdMYB9 and MdMYBPA1 to participate anthocyanin biosynthesis in red-fleshed apple

Anthocyanins are important secondary metabolites in plants. They are important for human health because of their antioxidant activities and because their dietary intake reduces the incidence of cardiovascular and cerebrovascular diseases and tumors. The biosynthesis of anthocyanins and its regulation in fruits and vegetables is a global research hotspot. Compared with cultivated apples, the red-fleshed apple is a relatively new and popular commodity in the market. Previous studies on red-fleshed apples have focused on the basis for the high anthocyanin content and the transcriptional regulation of anthocyanin synthesis. In the present study, we focused on the mechanism of microRNA-mediated post-transcriptional regulation of anthocyanin synthesis in red-fleshed apples. We identified a microRNA (miRNA), designated mdm-miR858, that is specifically expressed in the flesh of apple fruit. The expression level of miR858 was significantly lower in red-fleshed apples than in white-fleshed apples. The overexpression of mdm-miR858 significantly inhibited anthocyanin accumulation, whereas the silencing of mdm-miR858 promoted anthocyanin synthesis in STTM858 transgenic apple calli. Further analyses showed that mdm-miR858 targets the transcription factor genes MdMYB9 and MdMYBPA1 to participate anthocyanin accumulation in apple. Our results also show that MdHY5, a transcription factor in the light signaling pathway, can bind to the promoter of mdm-miR858 to inhibit its transcription, thereby regulating anthocyanin synthesis. Based on our results, we describe a novel HY5-miR858-MYB loop involved in the modulation of anthocyanin biosynthesis. These findings provide new information about how plant miRNAs regulate anthocyanin anabolism and provide a basis for breeding new anthocyanin-rich, red-fleshed apple varieties.