| The crabapples are one of the most important ornamental and economic germplasm resources in Malus Mill and Rosaceae. According to previous reports, anthocyanins, the plant secondary metabolites, are principally recognized for pigmenting to crabapple. In addition, its accumulation can improve the ornamental and medicinal value, as well as self-resistance in crabapple. Previously study showed that the E3 ubiquitin ligase COP1 (constitutively photomorphogenic 1) as a regulator involved in the anthocyanin synthesis and its gene promoter was possibly modified by DNA methylaiton via RdDM (RNA-directed DNA methylation) pathway. However, the underlying mechanism remains to be elucidated in crabapple. Thus, in this study, the crabapple cultivars ’Royalty’(both young and mature leaves are red to purple in color),’Flame’(both young and mature leaves are green), and ’Radiant’ (young leaves are red to purple and mature leaves are green) were used to analyze the mechanism that how the RdDM pathway methylate the promoter of McCOP1 and regulates anthocyanin biosynthesis.(1) Used of the DNA extracted from leaves samples of three crabapple cultivars as templates, the promoter sequence of McCOP1-a (2 195bp), McCOP1-b (2 195bp) and McCOP1-c (1 996bp) was cloned by hi-TAIL PCR methods.(2) Used of the total RNA extracted from leaves samples of ’Royalty’ cultivars as templates, the coding sequence was cloned by homologous cloning PCR. The genes contained an open reading frame of McCOP1-1 (1 941 bp), McCOP1-2 (1 872 bp), McAG04-A (2 736bp), McAGO4-Like (2 820bp), McDRM2 (1,803bp), McRDM1 (540bp) and encoded protein of 646,623,911,939,600,179 amino acids respectively.(3) DNA methylation and RT-qPCR assays showed that the expression levels of McCOP1 were suppressed after the promoter methylation occurred at Non-CG site and primarily asymmetric cytosine site CHH. To some extent, the gene expression level of McAGO4-A, McAGO4-Like, McDRM2, McRDM1 negatively correlated with McCOP1s.(4) It is demonstrated that the proteins McAGO4s and McDRM2 combined with McRDM1 to assemble complex to modify the McCOP1 promoter methylation by BiFC, Y1H and EMSA assays. Besides, the transcription factor McMYB10 was regulated by McCOP1.(5) The qRT-PCR and transgenic tobacco plants constitutively overexpressing assays showed that McANS plays an important role in the regulation of anthocyanin biosynthesisIn summary, these results suggested that the complex of McAGO4, McDRM2, and McRDM1 in RNA-directed DNA methylation pathway can methylated the McCOP1 promoter, leading to restrain levels of McCOP1 expression. As a result, the anthocyanins biosynthesis is advanced through regulating the accumulation of McMYB10 by McCOP1 protein. |
