PsbHLH1, a novel transcription factor involved in regulating anthocyanin biosynthesis in tree peony (Paeonia suffruticosa)

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.