Abstract
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 (McCOP1) from Malus cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the McCOP1 promoter was negatively correlated with McCOP1 RNA expression, and inhibiting DNA methylation caused decreased methylation of the McCOP1 promoter and asymmetric cytosine CHH methylation. We observed that the McCOP1 promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a McCOP1 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 McCOP1 promoter. Detection of CHH methylation and COP1 gene expression in the Arabidopsis thaliana atago4, atdrm2 and atrdm1 mutants showed that RDM1 is the effector of the RdDM pathway. This was confirmed by silencing McRDM1 in crabapple leaves or apple fruit, which resulted in a decrease in McCOP1 CHH methylation and an increase in McCOP1 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 McCOP1 promoter.
Highlights
Anthocyanins are flavonoid pigments that contribute much of the rich coloration in the plant kingdom, and they are involved in defense against biotic [1,2,3] and abiotic stresses [4]
A correlation analysis showed that the degree of CG, CHG and CHH methylation was negatively correlated with McCOP1-1 and McCOP1-2 expression, with correlation coefficients of −0.48, −0.43, −0.70 and −0.48, −0.42, −0.70, respectively in McCOP1-1 and McCOP1-2 (Figure 2D; Supplementary Table S2)
These results suggest that McCOP1 promoter methylation levels affect McCOP1 transcript levels
Summary
Anthocyanins are flavonoid pigments that contribute much of the rich coloration in the plant kingdom, and they are involved in defense against biotic [1,2,3] and abiotic stresses [4]. Anthocyanin biosynthesis is promoted by members of the large MYB transcription factor family, which recognize and bind to response cis-elements in the promoter regions of flavonoid biosynthesis genes [7,8,9,10,11,12]. Overexpression of pear (Pyrus communis) PbCOP1.1 was found to reduce red coloration in the fruit peel of the ‘Red Bartlett’ pear cultivar [18]. These findings further indicate that MdCOP1 is involved in ubiquitination and degradation of MdMYB1, thereby affecting anthocyanin biosynthesis. Crabapple represents an interesting experimental target to test for other potential regulatory systems that might influence anthocyanin accumulation, including potential epigenetic regulation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
