Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured ‘Fuji’ apples and found that differences in the transcript levels of MdMYB1 , which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway.

中文翻译：

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RdDM途径介导的MdMYB1基因座的甲基化调节苹果花色苷的生物合成。

据报道，苹果运动中MdMYB1启动子的甲基化是花青素途径的调节剂，但是对于甲基化机制如何识别该位点以调节花青素积累的了解很少。在这项研究中，我们分析了三种颜色不同的“富士”苹果，发现MdMYB1的转录水平差异（编码花色苷生物合成的关键调节剂）控制了果皮中花色苷的含量（因此，颜色）。发现MdMYB1启动子的MR3区（-1246至-780）中的CHH甲基化水平与MdMYB1负相关。表达。因此，它们是研究苹果运动中DNA甲基化的理想材料。发现负责CHH甲基化的RNA定向DNA甲基化（RdDM）途径蛋白MdAGO4与MdMYB1启动子相互作用。MdAGO4可与MdRDM1和MdDRM2相互作用形成效应子复合物，从而实现CHH甲基化。当MdAGO4和MdDRM2在苹果愈伤组织和拟南芥突变体中过表达时，这些蛋白质会增加AGO4结合位点的CHH甲基化。在电泳迁移率变动分析中，发现MdAGO4与含有ATATCAGA的序列特异性结合。击倒MdNRPE1不会影响MdAGO4与MdMYB1的c3区域的结合。35S :: AGO4愈伤组织的启动子。两者合计，我们的数据表明，MdMYB1基因座通过MdAGO4与MdMYB1启动子的结合而甲基化，从而通过RdDM途径调节花色苷的生物合成。