Identification and functional characterization of AmbHLH002 as a conserved bHLH regulator of anthocyanin biosynthesis in Antirrhinum majus

Basic helix-loop-helix (bHLH) transcription factors regulate diverse plant processes, particularly anthocyanin biosynthesis through the MYB-bHLH-WD40 complex. Despite snapdragon (Antirrhinum majus) serving as a classical model for studying flower pigmentation genetics, its bHLH gene family has rarely been comprehensively characterized. Here, we performed a genome-wide identification and systematic characterization of the bHLH gene family in A. majus, with a focus on candidates involved in anthocyanin biosynthesis. A total of 150 AmbHLH genes were identified and subjected to in-silico analyses, including phylogenetic classification, structural analysis, and promoter cis-element characterization. Comparative transcriptomic profiling between anthocyanin-poor (“SIPPE50”, Green) and anthocyanin-rich (“JI2R”, Red) snapdragon lines highlighted eight differentially expressed AmbHLHs. AmbHLH001, AmbHLH002, and AmbHLH042 showed significant upregulation in the anthocyanin-rich line and showed positive correlations with the expression of key anthocyanin biosynthetic genes. Among these, AmbHLH002 was prioritized as a candidate and was assessed via heterologous overexpression in tomatoes. Notably, AmbHLH002 is a newly identified regulator whose overexpression in tomato resulted in visible purple pigmentation and increased anthocyanin accumulation. These findings support the view that AmbHLH002 acts as a positive regulator, with phylogenetic evidence for conservation of anthocyanin biosynthesis, presenting valuable potential for engineering pigmentation traits in ornamental plants and serving as a candidate visible marker for plant genetic transformation.