The blood red color of pigmented orange fruit varieties [Citrus sinensis L. (Osbeck)] is due to the presence of anthocyanin pigments that largely contribute to determine the high organoleptic qualities and the nutritional properties of the fruits. The content of pigments in sweet orange depends primarily on genetic factors and on environmental conditions. In particular, it has been extensively shown that cold temperature induces an increase of anthocyanin content that is achieved by the induction of the related gene expression. The purpose of our work is to understand the mechanism underlying the color variegation occurring inside the blood oranges during the cold induction of anthocyanin biosynthesis, despite the fact that the entire fruit is genotypically programmed to produce pigments. Therefore, the amount of anthocyanin and the expression of both structural and regulatory genes have been monitored in either high-pigmented (HP) or not/low pigmented (NP) segments of the same fruit during the storage at 4 °C for a total experimental period of 25 days. Our results clearly indicate that the anthocyanin content is directly correlated with the levels of gene transcription, with higher pigmented areas showing higher enhancement of gene expression. Furthermore, we analyzed the reshaping of the DNA methylation status at the promoter regions of genes related to anthocyanin biosynthetic pathway, such as DFR and Ruby. Our results unequivocally demonstrate that in the promoter regions of both DFR and Ruby, the amount of cytosine methylation strongly decreases along the cold storage in the HP areas, whereas it increases in the NP areas of the same fruit, probably causing a partial block of the gene transcription. Finally, by measuring the changes in the expression levels of the Citrus DNA demethylases, we found that DML1 might play a crucial role in determining the observed demethylation of DFR and Ruby promoters, with its expression induced by cold in the HP areas of the fruits. This is the first report in which different levels of gene expression implicated in anthocyanin production in blood orange fruit is correlated with an epigenetic control mechanism such as promoter methylation.

中文翻译：

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冷胁迫下甜橙果实[Citrus sinensis L.（Osbeck）]的花色苷生物合成和DNA甲基化动力学。

有色橙色水果品种[柑橘]的血红色L.（Osbeck）]是由于花色苷色素的存在极大地决定了水果的高感官品质和营养特性。甜橙中色素的含量主要取决于遗传因素和环境条件。特别地，已经广泛地表明，低温诱导花青素含量的增加，这是通过诱导相关基因表达实现的。我们的工作目的是了解在冷诱导花青素生物合成过程中血橙内部发生颜色变化的机理，尽管事实上整个水果都经过了基因分型编程以产生色素。因此，在4°C的温度下进行了整个实验期间，在同一水果的高色素（HP）或无色素/低色素（NP）片段中，花色苷的量以及结构和调节基因的表达都得到了监测。 25天 我们的结果清楚地表明，花色苷含量与基因转录水平直接相关，色素较高的区域显示出基因表达的增强。此外，我们分析了与花色苷生物合成途径有关的基因的启动子区域DNA甲基化状态的重塑，例如 我们的结果清楚地表明，花色苷含量与基因转录水平直接相关，色素较高的区域显示出基因表达的增强。此外，我们分析了与花色苷生物合成途径有关的基因的启动子区域DNA甲基化状态的重塑，例如 我们的结果清楚地表明，花色苷含量与基因转录水平直接相关，色素较高的区域显示出基因表达的增强。此外，我们分析了与花色苷生物合成途径有关的基因的启动子区域DNA甲基化状态的重塑，例如DFR和Ruby。我们的结果清楚地表明，在DFR和Ruby的启动子区域，胞嘧啶甲基化的数量沿HP区域的冷藏显着减少，而在同一果实的NP区域则增加，可能导致部分果糖的阻塞。基因转录。最后，通过测量柑橘DNA脱甲基酶表达水平的变化，我们发现DML1可能在确定观察到的DFR和Ruby脱甲基中起关键作用启动子，其表达在果实的HP区受冷诱导。这是第一份报道，其中涉及血橙果实中花色苷生产的不同水平的基因表达与表观遗传控制机制（如启动子甲基化）相关。