MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple

Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four continuous-generation bud sport mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin color traits remains elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in 'Golden Delicious' fruits, 'Gala' calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in 'Red Fuji' apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.