Genome-Wide Identification of the bHLH Gene Family in Reveals Its Potential Role in the Regulation of Isoflavonoid Biosynthesis.

(Champ. ex Benth.) Schot is a significant leguminous plant valued for its edible tuberous roots, which are a plentiful source of isoflavonoids. Basic helix-loop-helix (bHLH) transcription factors (TFs) have been reported to regulate secondary metabolism in plants, especially flavonoid biosynthesis. However, the genes in have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 genes were identified in the genome, classifying them into 23 subfamilies based on the gene structures and phylogenetic relationships. All the CsbHLH proteins contained both motifs 1 and 2, whereas motif 8 was only distributed in subgroup III (d + e). Collinearity analysis demonstrated that fragmental replications are the primary driver of CsbHLH evolution, with the majority of duplicated gene pairs experiencing selective pressure. Nine candidate genes were found to play a potential role in regulating isoflavonoid biosynthesis through a combination of gene-to-metabolite correlation analysis and weighted gene co-expression network analysis (WGCNA). Additionally, the -regulatory elements and response to MeJA of these nine genes were characterized and confirmed through quantitative real-time PCR (qRT-PCR) analysis. Among them, three CsbHLHs (CsbHLH9, CsbHLH89, and CsbHLH95) were selected for further investigation. Yeast two-hybrid (Y2H), dual-luciferase (LUC) assays, bimolecular fluorescence complementation (BiFC) assays, and transient transformation demonstrated that CsbHLH9 acted as a transcriptional activator through its interaction with CsMYB36 and binding to the promoters of isoflavonoid biosynthesis genes in a MeJA-induced manner, such as , , and , to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in .