Flower color is one of the most important features of ornamental plants. Anthocyanin composition and concentration are usually closely related to flower color formation. The biosynthesis of anthocyanin is regulated by a series of structural genes and regulatory genes. The basic helix-loop-helix proteins (bHLHs) are considered as one of the key transcription factors known as the regulators of anthocyanin biosynthesis. However, the bHLH transcription factor family of tree peony (Paeonia suffruticosa) has not been systematically studied in previous studies, especially for the regulation of petal pigmentation. The aim of this study was to identify bHLH genes and unravel their underlying molecular mechanism involved in the regulation of anthocyanin biosynthesis in tree peony. Based on transcriptome profiling analysis, we identified three bHLHs candidate anthocyanin regulators, PsbHLH1, PsbHLH2, and PsbHLH3. PsbHLH1-3 were phylogenetically clustered in the IIIf bHLH subgroup, which is involved in anthocyanin biosynthesis in other plant species. In addition, three bHLH proteins were localized in the nucleus and displayed transcriptional activation activity in a yeast hybrid system. Through a series of functional experiments, we further demonstrated that PsbHLH1 could transcriptionally activate the expression of PsDFR and PsANS via directly binding to their promoters. These results laid a solid foundation to better understand the regulatory mechanisms of anthocyanin biosynthesis in P. suffruticosa and to benefit molecular breeding of tree peony cultivars with novel color.

花的颜色是观赏植物的最重要特征之一。花色苷的组成和浓度通常与花的颜色形成密切相关。花色苷的生物合成受到一系列结构基因和调控基因的调控。基本的螺旋-环-螺旋蛋白（bHLH）被认为是被称为花色苷生物合成调节剂的关键转录因子之一。然而，在先前的研究中，还没有系统地研究牡丹（bpahonia suffruticosa）的bHLH转录因子家族，特别是在调节花瓣色素沉着方面。这项研究的目的是鉴定bHLH基因并揭示其参与调控牡丹花色苷生物合成的潜在分子机制。基于转录组分析，我们确定了三种bHLHs候选花色苷调节剂PsbHLH1，PsbHLH2和PsbHLH3。PsbHLH1-3在系统发育上聚集在IIIf bHLH亚组中，该亚组参与其他植物物种中的花色苷生物合成。此外，三种bHLH蛋白位于核内，并在酵母杂交系统中表现出转录激活活性。通过一系列功能实验，我们进一步证明了PsbHLH1可以转录激活PsDFR的表达通过直接与其启动子结合而形成PsANS。这些结果为更好地了解P. suffruticosa花色苷生物合成的调控机制和有益于新颖颜色的牡丹品种分子育种奠定了坚实的基础。