Genome-Wide Identification of bHLH Transcription Factors and Screening of Candidate Regulators Associated with Flavonoid Biosynthesis in Platycodon grandiflorus Under Methyl Jasmonate Treatment.

Abstract

Background: The bHLH transcription factor family plays crucial roles in plant secondary metabolism, yet its genome-wide characterization and regulatory functions in flavonoid biosynthesis remain unexplored in Platycodon grandiflorus, a medicinal species with bioactive flavonoids. This study aims to systematically identify PgbHLH genes and screen for candidate regulators of flavonoid production. Methods: We performed genome-wide identification of bHLH family members using curated reference genomes, followed by phylogenetic classification, chromosomal localization, gene structure analysis, and collinearity assessment. Promoter cis-element prediction and subcellular localization assays for selected proteins were conducted. Expression profiles under methyl jasmonate (MeJA) treatment were evaluated via transcriptome sequencing and RT-qPCR. Protein-protein interaction prediction, expression correlation, and weighted gene co-expression network analysis (WGCNA) were integrated to link PgbHLH genes with flavonoid structural genes. Results: A total of 95 PgbHLH genes were identified and clustered into 23 subfamilies, unevenly distributed across nine chromosomes. All members retained conserved bHLH domains but varied in exon-intron structures. Thirty-six duplication events were detected, with Ka/Ks ratios indicating purifying selection. Promoters harbored multiple hormone- and stress-responsive elements. PgbHLH37, PgbHLH42, and PgbHLH70 were confirmed to be nuclear-localized. Transcriptomic and RT-qPCR data showed differential expression of PgbHLH genes in response to MeJA. Integrative analyses identified 23 PgbHLH members associated with flavonoid biosynthetic genes, among which PgbHLH37, PgbHLH42, and PgbHLH70 exhibited strong multi-dimensional correlations and were nominated as high-priority candidate regulators. Conclusions: This systematic characterization provides a foundation for understanding bHLH-mediated flavonoid metabolism in P. grandiflorus, with three prioritized candidates for future functional validation. The findings are strictly supported by the presented genomic and expression evidence. However, our conclusions are based on total flavonoid content, and analysis of individual flavonoid species is required to determine pathway-specific regulation.