The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple ( cv. spp.) Leaves by Methylating the Promoter.

The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 () from cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the promoter was negatively correlated with RNA expression, and inhibiting DNA methylation caused decreased methylation of the promoter and asymmetric cytosine CHH methylation. We observed that the promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the promoter. Detection of CHH methylation and COP1 gene expression in the , and mutants showed that is the effector of the RdDM pathway. This was confirmed by silencing in crabapple leaves or apple fruit, which resulted in a decrease in CHH methylation and an increase in transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the promoter.