Convergent roles of BcGUN4.1, BcSG1, BcCHLH, and BcTPR4 in regulating the leaf greenness of non-heading Chinese cabbage

Abstract Non-heading Chinese cabbage (NHCC) extensively cultivated as a leafy vegetable in China, displays diverse leaf coloration, with green being an essential trait. Leaf greenness, determined by chlorophyll and carotenoid contents, is a crucial visual characteristic, and understanding its genetic and molecular mechanisms is essential for breeding. This study identified two highly homologous BcGUN4 genes ( BcGUN4.1 and BcGUN4.2 ) in NHCC. BcGUN4.1 exhibited localization to chloroplasts and cell membranes, positively regulating pigment accumulation. Similarly, BcSG1 and BcCHLH were detected in chloroplasts and cell membranes, co-localizing with BcGUN4.1. Notably, BcGUN4.1 interacted with BcSG1 and BcCHLH in chloroplasts, while BcSG1 functioned as a positive regulator of pigmentation. Moreover, BcGUN4.1, BcSG1, and BcCHLH interacted with BcTPR4, and BcSG1 directly bound to BcCHLH, forming a tetrameric complex in chloroplasts. BcTPR4 contributed to pigment accumulation. Additionally, BcSG1 and BcTPR4 enhanced BcGUN4.1–BcCHLH complex activity. These findings established that BcGUN4.1, BcSG1, BcCHLH, and BcTPR4 cooperatively regulate leaf greenness in NHCC, offering novel insights into the molecular mechanisms underlying this trait.