Overexpression of CpbHLH1 in Arabidopsis and tobacco resulted in a dramatic decrease in anthocyanin accumulation by repressing the expression of late biosynthesis genes in the flavonoid biosynthesis pathway. Many basic helix-loop-helix (bHLH) transcription factors (TFs) of subgroup IIIf have been characterized as anthocyanin-associated activators in higher plants, but information regarding bHLH TFs that inhibit anthocyanin accumulation remains scarce. In this study, the subgroup IIIf bHLH TF CpbHLH1 from Chimonanthus praecox (L.) was identified as a negative regulator of anthocyanin accumulation. Our results showed that overexpression of CpbHLH1 in model plant species, Arabidopsis and tobacco, resulted in a dramatic decrease in anthocyanin content, whereas the content of proanthocyanidin was little affected. Quantitative RT-PCR (qRT-PCR) assays of the structural genes in the flavonoid biosynthesis pathway revealed that CpbHLH1 inhibits anthocyanin accumulation mainly through repressing the expression of late biosynthesis genes (LBGs). Interactions between CpbHLH1 protein and AtPAP1/NtAN2 protein were detected via yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. This is the first bHLH repressor of anthocyanin biosynthesis identified in dicotyledons. These results can help us better understand the anthocyanin regulatory network in plants and may provide insights into the diverse functions of bHLH proteins.

中文翻译：

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来自蜡梅 (L.) 的亚组 IIIf bHLH 转录因子 CpbHLH1 在转基因模型植物中的表达抑制花青素积累。


拟南芥和烟草中 CpbHLH1 的过度表达通过抑制类黄酮生物合成途径中晚期生物合成基因的表达，导致花青素积累急剧减少。许多 IIIf 亚组的碱性螺旋-环-螺旋 (bHLH) 转录因子 (TF) 已被定性为高等植物中的花青素相关激活剂，但有关抑制花青素积累的 bHLH 转录因子 (TF) 的信息仍然很少。在这项研究中，来自腊梅 (L.) 的 IIIf bHLH TF CpbHLH1 亚组被确定为花青素积累的负调节因子。我们的结果表明，模式植物拟南芥和烟草中CpbHLH1的过度表达导致花青素含量急剧下降，而原花青素含量几乎没有受到影响。对类黄酮生物合成途径中结构基因的定量 RT-PCR (qRT-PCR) 分析表明，CpbHLH1 主要通过抑制晚期生物合成基因 (LBG) 的表达来抑制花青素积累。通过酵母双杂交 (Y2H) 和双分子荧光互补 (BiFC) 测定检测 CpbHLH1 蛋白和 AtPAP1/NtAN2 蛋白之间的相互作用。这是在双子叶植物中发现的第一个花青素生物合成的 bHLH 阻遏蛋白。这些结果可以帮助我们更好地了解植物中的花青素调节网络，并可能为bHLH蛋白的多种功能提供见解。