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

<i>Callerya speciosa</i> (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 <i>bHLH</i> genes in <i>C. speciosa</i> have not yet been reported, and their regulatory role in isoflavonoid biosynthesis remains unexplored. Here, 146 <i>CsbHLH</i> genes were identified in the <i>C. speciosa</i> 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 <i>CsbHLH</i> gene pairs experiencing selective pressure. Nine candidate <i>CsbHLH</i> 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 <i>cis</i>-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 <i>CsIFR2</i>, <i>CsI3'H2</i>, and <i>CsCHS4</i>, to promote isoflavonoid (calycosin, calycosin-7-o-glucoside, and formononetin) accumulation. Our results establish a basis for the functional analysis of <i>bHLH</i> genes and investigations into the molecular mechanisms underlying isoflavonoid biosynthesis in <i>C. speciosa</i>.