Identification of TPS-d subfamily genes and functional characterization of three monoterpene synthases in slash pine

Oleoresin terpenoids have been widely used in the food, pharmaceutical, and chemical industries. A thorough understanding of oleoresin terpenoid biosynthesis is important for the metabolic engineering of high-valued terpenoid products in plants and microbes and the improvement of oleoresin traits using molecular breeding strategies. TPS-d subfamily members, specific to conifers, determine the oleoresin terpenoid compound diversity. Here, we aimed to identify the TPS-d genes in slash pine (Pinus elliottii) and characterize their function and expression patterns in population. Our study reveals two main results: (1) A total of 52 terpene synthase (TPS) candidate genes were identified based on long-read transcripts of slash pine, 12 of which were successfully cloned from the secondary developing xylem RNA. Two levopimaradiene/abietadiene synthases (LAS), one monofunctional isopimaradiene synthase (mISO1)-like, and two alpha-pinene synthases were identified by comparing with previously known TPS. Next, the two alpha-pinene synthases and one beta-pinene synthase producing multiple products were validated through heterologous expression and in vitro enzyme activity assays, which showed different product profiles from previously identified TPS. (2) The expression status of the cloned TPS genes in 240 slash pine individuals were analyzed. Four exhibited very low expression levels, including those identical to copalyl diphosphate synthases, hemiterpene synthases, diterpene synthases without activity, and one obtained without soluble protein in the heterologous expression. The expression levels of eight TPS genes participating in slash pine oleoresin biosynthesis varied in different individuals and were highly correlated with multiple transcriptional regulators, namely, MYB and MYC. The expression of two alpha-pinene synthase genes was significantly higher than that of the other TPS genes. Some TPS gene expression levels were positively correlated with the corresponding oleoresin component content. Our results provide an essential reference for exploring the mechanisms of terpene biosynthesis and regulation.