Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar ‘Daihong’ (‘DH’) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5’ and 3’ flanking regions of genes and transposable elements (TEs), and in TE body in all CG, CHG and CHH context, especially mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1 and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin, respectively. The treatment of DNA demethylation agent 5-aza-2’-deoxycitydine verified the negative correlation between DNA methylation and gene expression within flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during ‘DH’ apple development.

中文翻译：

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类黄酮途径基因启动子区的高甲基化与“大红”苹果果实发育过程中的肤色褪色有关

尽管苹果果皮颜色形成的分子机制众所周知，但人们对苹果果皮颜色褪色的了解还不是很清楚。红肉苹果品种“大红”（“DH”）尽管具有杂合 R6 等位基因，但缺乏红色果皮的 Red-TE，但在果实发育过程中表现出果皮颜色褪色。在这项研究中，转录组分析显示，MdMYB10 的表达水平随着果实发育而增加，而观察到 MdMYBPA1、MdCHS、MdANS、MdUFGT、MdLAR 和 MdANR 的表达水平降低，这与查尔酮、花青素、儿茶素、表儿茶素和原花青素B2。全基因组亚硫酸氢盐测序 (WGBS) 表明胞嘧啶甲基化水平整体增加，基因和转座元件 (TE) 的 5' 和 3' 侧翼区域以及所有 CG、CHG 和 CHH 背景下的 TE 体甲基化增加，尤其是mCHH 背景，在果实发育过程中。DNA 甲基化增加归因于 DNA 去甲基化酶基因（包括 MdDME1、MdROS1 和 MdROS2）表达水平降低。关联分析显示MdCHS、MdCHI、MdMYBPA1启动子甲基化水平与其各自的转录本水平呈显着负相关，MdCHS、MdCHI、MdANR、MdFLS启动子甲基化水平与查耳酮、柚皮素含量呈显着负相关。分别是-7-葡萄糖苷、表儿茶素和槲皮素。DNA去甲基化剂5-aza-2'-deoxycydine的处理证实了类黄酮途径中DNA甲基化与基因表达之间的负相关性。这些发现表明，类黄酮生物合成途径基因启动子区域的高甲基化与“DH”苹果发育过程中基因表达和类黄酮含量的减少以及果实表皮颜色褪色有关。