Anthocyanins are important metabolites that provide a red or blue–purple hue to plants. The biosynthesis of these metabolites is mainly activated by the MYB-bHLH-WD40 (MBW) complex and repressed by a wide variety of proteins. Studies have shown that MYB activators activate MYB repressors to balance anthocyanin biosynthesis. However, there is a scarcity of studies investigating this mechanism in grapes. To explore the transcription factors involved in the regulation of anthocyanin biosynthesis, we reanalyzed the RNA-seq database for different developmental stages of ‘Muscat Hamburg’ berries, and the R2R3-MYB gene, annotated as VvMYB3, was screened. Our study revealed the anthocyanin content of the grape cultivar ‘Y73’ was higher than that of its parental cultivar MH, and the putative repressor VvMYB3 was found to be highly expressed in ‘Y73’ by qRT-PCR. The calli transgenic assays demonstrated that the repressive activity of VvMYB3 was conferred by the bHLH-binding motif, as well as by the C1 and C2 motifs. Yeast hybridization and chip-PCR assays revealed that VvMYB3 could repress anthocyanin biosynthesis by competing with VvMYBA1 to bind to VvMYC1 and promoting histone deacetylation of VvUFGT via the C2 motif. However, the expression of VvMYB3 was activated by VvMYBA1, which forms a negative feedback regulatory loop to modulate anthocyanin accumulation. In addition, we found a 408-bp repeat tandem sequence insertion in the VvMYBA1 promoter region of ‘Y73’ by sequencing. The GUS activity analysis showed that this sequence enhanced the expression of VvMYBA1 and led to an excessive accumulation of anthocyanins. Overall, our results provide insights into the anthocyanin activator–repressor system in grapes that prevents overaccumulation of anthocyanins.

中文翻译：

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VvMYBA1和VvMYB3形成激活-阻遏系统来调节葡萄中花​​青素的生物合成


花青素是重要的代谢物，为植物提供红色或蓝紫色色调。这些代谢物的生物合成主要由 MYB-bHLH-WD40 (MBW) 复合物激活，并受到多种蛋白质的抑制。研究表明，MYB 激活剂可激活 MYB 阻抑剂，以平衡花青素生物合成。然而，很少有研究调查葡萄中的这一机制。为了探索花青素生物合成调控中涉及的转录因子，我们重新分析了‘Muscat Hamburg’浆果不同发育阶段的RNA-seq数据库，并筛选了R2R3-MYB基因，注释为VvMYB3。我们的研究表明，葡萄品种“Y73”的花青素含量高于其亲本品种 MH，并且通过 qRT-PCR 发现假定的阻遏蛋白 VvMYB3 在“Y73”中高表达。愈伤组织转基因测定表明，VvMYB3 的抑制活性是由 bHLH 结合基序以及 C1 和 C2 基序赋予的。酵母杂交和芯片 PCR 检测表明，VvMYB3 可以通过与 VvMYBA1 竞争结合 VvMYC1 并通过 C2 基序促进 VvUFGT 的组蛋白脱乙酰化来抑制花青素生物合成。然而，VvMYB3的表达被VvMYBA1激活，形成负反馈调节环来调节花青素的积累。此外，我们通过测序发现“Y73”的 VvMYBA1 启动子区域有一个 408 bp 的重复串联序列插入。 GUS活性分析表明该序列增强了VvMYBA1的表达并导致花青素的过度积累。总的来说，我们的研究结果提供了对葡萄中花青素激活剂-阻遏剂系统的深入了解，该系统可防止花青素过度积累。