Wintersweet (Chimonanthus praecox (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 C. praecox bHLH (CpbHLH) gene family, identifying a total of 131 CpbHLH genes across 11 chromosomes. Phylogenetic analysis classified these CpbHLH genes into 23 subfamilies, wherein most members within the same subfamily exhibited analogous intron/exon patterns and motif composition. Moreover, the expansion of the CpbHLH gene family was primarily driven by segmental duplication, with duplicated gene pairs experiencing purifying selection during evolution. Transcriptomic analysis revealed diverse expression patterns of CpbHLH 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 CpbHLH gene family, laying a solid foundation for further explorations of the multifaceted physiological and molecular roles of bHLH TFs in Wintersweet.

Terpene synthase (TPS) plays a key role in the biosynthesis of terpenoids, which are the most important components of the volatile compounds of wintersweet (Chimonanthus praecox). In this study, 52 CpTPS genes were found in wintersweet which were divided into 5 subfamilies. We identified 146 SSRs in the CpTPS genes, and obtained 33 pairs of SSR primers with good polymorphism through amplification in 6 wintersweet samples. Then, these primers were amplified in 69 samples from China’s main wintersweet production areas. Through structural analysis, 69 samples were divided into 2 clusters, and were divided into 4 groups in a genetic cluster analysis, of which SH-33 and SW were separate groups. Through AMOVA analysis, it was found that the variation mainly occurred in the population, and that the gene flow between populations was Nm > 1, so it might lead to population differentiation. In other words, these findings provided useful information for the biosynthesis of terpenoids, the construction of a genetic linkage map, the detection of quantitative trait loci, marker-assisted selection and other aspects of wintersweet.

Wintersweet (Chimonanthus praecox) is known for its flowering in winter and its rich floral aroma; the whole flower is yellow and the inner petals are red. In this study, we chose the wintersweet genotypes HLT040 and HLT015 as the research materials, and studied the co-regulatory mechanism of color and fragrance of wintersweet through metabolomics and transcriptomics. This study found that there were more flavonoids in HLT015, and anthocyanins (cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside) were only present in HLT015, but HLT040 contained more monoterpenes and FVBPs (phenylpropanoid volatile compounds) than HLT015. We constructed putative benzenoids and phenylpropanoid metabolism pathway as well as terpene metabolism pathways. We found some linkages between the different structural genes and metabolites for flower color and fragrance in wintersweet, and screened out 39 TFs that may be related to one or more structural genes in benzenoids and phenylpropanoid or terpene metabolism pathways. In the yeast one-hybrid assay, we found that CpERF7 was able to interact with the promoter of CpANS1, while CpbHLH50 and CpMYB21 interacted with the promoter of CpTPS4. This study provides a theoretical basis for understanding the co-regulatory mechanism of color and fragrance in wintersweet.

Wintersweet (Chimonanthus praecox) is known for its flowering in winter and rich floral aroma, and it’s the whole flower is yellow or the inner petals are red. In this study, we choose the wintersweet genotypes HLT040 and HLT015 (belonging to the C. praecox 'Luteus' group and C. praecox 'Grandiflorus' group, respectively) as the re-search materials, and studied the co-regulatory mechanism of color and fragrance of wintersweet through metabolomics and transcriptomics. This study found that there were more flavonoids in HLT015, and anthocyanins (Cyanidin-3-O-rutinoside and Cyanidin-3-O-glucoside) were only present in HLT015, but HLT040 contained more monoterpenes and FVBPs (phenylpropanoid volatile compounds) than HLT015. We constructed putative benzenoids and phenylpropanoid metabolism pathway as well as terpene metabolism pathways. We found some linkages between the different struc-tural genes and metabolites in flower color and fragrance of wintersweet, and screened out 34 TFs that may be related to one or more structural genes in benzenoids and phenylpropanoid or terpene metabolism pathways. This study provided a theo-retical basis for understanding the co-regulatory mechanism of color and fragrance in wintersweet.