Xing Yifan, Xie Ziyi, Sun Weilei, Sun Yuying, Han Zhenyun, Zhang Shiya, Tian Ji, Zhang Jie, Yao Yuncong
Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing University of Agriculture, Beijing 102206, China.
Department of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China.
Plants (Basel). 2021 Nov 15;10(11):2466. doi: 10.3390/plants10112466.
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.
已知植物中花青素色素的合成受多种机制调控，包括表观遗传调控；然而，RNA 介导的 DNA 甲基化（RdDM）途径的作用尚不清楚。在这里，我们使用亚硫酸氢盐测序和实时（RT）定量（q）PCR 分析了来自 cv. spp 的组成型光形态建成 1（）启动子的甲基化水平，该基因参与调控花青素生物合成。启动子的 CHH 甲基化水平与 RNA 表达呈负相关，抑制 DNA 甲基化导致启动子甲基化减少和不对称胞嘧啶 CHH 甲基化。我们观察到启动子是 RdDM 途径的 Argonaute RISC 组分 4（McAGO4）蛋白的直接靶点，该蛋白与启动子的 GGTTCGG 位点结合。双分子荧光互补（BIFC）分析表明，RNA 介导的 DNA 甲基化（McRDM1）与 McAGO4 和另一种 RdDM 蛋白结构域重排甲基转移酶 2（McDRM2）相互作用，以调节启动子的 CHH 甲基化。对、和突变体中 CHH 甲基化和 COP1 基因表达的检测表明，是 RdDM 途径的效应器。通过在海棠叶或苹果果实中沉默来证实这一点，这导致 CHH 甲基化减少，转录水平增加，以及花青素积累增加。总之，这些结果表明 RdDM 途径通过启动子的 CHH 甲基化参与调控花青素积累。
