Evolution of Terpene Synthases in the Sesquiterpene Biosynthesis Pathway and Analysis of Their Transcriptional Regulatory Network in Asteraceae

Abstract The Asteraceae family, one of the largest angiosperm families, is rich in terpenoid secondary metabolites with significant medicinal value. Asteraceae plants have evolved a diverse array of terpenoid biosynthesis pathways, reflecting their adaptive significance and complex regulatory mechanisms. However, the evolutionary patterns and transcriptional regulatory mechanisms governing these biosynthetic processes remain unclear. This study investigates the evolution and transcriptional regulation of terpenoid biosynthesis genes in Asteraceae. Comparative genomic analysis of 19 Asteraceae and six out-group species revealed that Asteraceae species diverged ~74.03 million years ago and were distinctly divided into three sub-families. A total of 1,714 TPS genes were identified, predominantly in the TPS-a and TPS-b subfamilies. Caryophyllene-type sesquiterpene biosynthetic gene clusters (BGCs) were detected in 10 species, with their formation due to whole-genome duplication (WGD) and tandem duplication. By integrating weighted gene co-expression network analysis (WGCNA) and machine learning methods, key transcription factors regulating caryophyllene synthase (CPS) in Carthamus tinctorius were identified. A multi- layered gene regulatory network was constructed to identify potential regulatory factors involved in TPS gene regulation under light stress. By exploring the evolutionary patterns and potential regulatory relationships involved in terpenoid biosynthesis in Asteraceae, this study provides important insights into TPS gene evolution. In addition, the findings also offer guidance for optimizing genetic engineering strategies in terpenoid-based drug development.