Abstract

Tulips are globally renowned for their ornamental value. Colors of tulips are critical attributes, with the rare black tulip tepal pigmentation being particularly linked to the biosynthesis of anthocyanins. Transcriptomic analysis and functional assays revealed that TgbHLH1 directly bound to the TgANS promoter, identifying it as a key positive regulator of anthocyanin biosynthesis in ‘Queen of Night’. We further identified two complexes (TgbHLH1-TgMYB1 and TgbHLH1-TgMYB2) associated with anthocyanin biosynthesis. The interaction between TgbHLH1 and TgMYB1 resulted in an observable accumulation of anthocyanins after the budding stage. During these stages, the TgMYB1-TgbHLH1 complex enhanced the transcriptional capacity of TgbHLH1 to upregulate the anthocyanin biosynthesis. During the green bud stage, the negative regulator TgMYB2 repressed the transcriptional capacity of TgbHLH1 by interacting with TgbHLH1. Taken together, our results demonstrated TgbHLH1 formed two complexes with TgMYB1 and TgMYB2 to balance anthocyanin biosynthesis during floral development. The regulatory network provides a new mechanistic explanation for black tulip tepal pigmentation, and prepares for genetic breeding of tulips with more valuable cultivars.