Comparative genomic analysis of bHLH genes across multiple species uncovers a novel gene regulating ginsenoside biosynthesis in Panax ginseng

Basic helix–loop–helix (bHLH) transcription factors are the second largest transcription factor family in plants and regulate diverse physiological processes. This study aimed to investigate the evolution, expansion, and functional divergence of bHLH genes in Panax species and to identify new gene involved in ginsenoside biosynthesis. bHLH genes from six species (Daucus carota, Eleutherococcus senticosus, Panax notoginseng, Panax vietnamensis, Panax quinquefolius, and Panax ginseng) were identified using genome-wide screening. Phylogenetic analysis, inter-species synteny, and structure comparison were performed. Subcellular localization, dual-luciferase reporter assays, and in vivo functional validation were conducted for target bHLH gene from Panax ginseng. A total of 1,254 bHLH genes were identified and classified into 17 subfamilies. Differences in gene numbers suggested that whole-genome duplication events contributed to family expansion. Synteny analysis revealed 49 orthologous bHLH gene units, most of which showed variation in intron number, potentially leading to functional divergence. Nuclear-localized protein encoded by the sole Myelocytomatosis (MYC) gene Pg.bHLH203 among the orthologous bHLH genes, regulates ginsenoside biosynthesis by modulating the transcription of the key enzyme genes Pg.DS and Pg.PPDS in the ginsenoside biosynthetic pathway. These results suggest that Pg.bHLH203 is a novel regulator of ginsenoside biosynthesis, identified through evolutionary insights and confirmed by functional validation. This study provides the first comprehensive analysis of bHLH gene evolution in Panax species, filling a key knowledge gap, advancing the understanding of the evolutionary trajectory of this important transcription factor family.