Genome-Wide Identification and Expression Analysis of the BHLH Transcription Factor Family in Wintersweet ()

Wintersweet ( (L.) Link, Calycanthaceae) is an esteemed ornamental flowering shrub known for its distinct blooming period in winter, vibrant color petals, and captivating floral fragrance. Basic helix-loop-helix (bHLH) transcription factors (TFs) play pivotal roles as key regulators in secondary metabolites biosynthesis, growth, and development in plants. However, the systematic analysis of the bHLH family members and their role in the regulation of floral traits in Wintersweet remains insufficiently understood. To bridge this knowledge gap, we conducted a comprehensive genome-wide analysis of the bHLH () gene family, identifying a total of 131 genes across 11 chromosomes. Phylogenetic analysis classified these genes into 23 subfamilies, wherein most members within the same subfamily exhibited analogous intron/exon patterns and motif composition. Moreover, the expansion of the gene family was primarily driven by segmental duplication, with duplicated gene pairs experiencing purifying selection during evolution. Transcriptomic analysis revealed diverse expression patterns of genes in various tissues and distinct stages of Wintersweet flower development, thereby suggesting their involvement in a diverse array of physiological processes. Furthermore, yeast 2-hybrid assay demonstrated interaction between CpbHLH25 and CpbHLH59 (regulators of floral scent and color) as well as with CpbHLH112 and CpMYB2, suggesting potential coordinately regulation of secondary metabolites biosynthesis in Wintersweet flowers. Collectively, our comprehensive analysis provides valuable insights into the structural attributes, evolutionary dynamics, and expression profiles of the gene family, laying a solid foundation for further explorations of the multifaceted physiological and molecular roles of bHLH TFs in Wintersweet.