Divergent Evolution of the Diterpene Biosynthesis Pathway in Tea Plants (Camellia sinensis) Caused by Single Amino Acid Variation of ent-Kaurene Synthase

Most plant terpenoids are classified as secondary metabolites. A small portion of them are products of primary metabolism biosynthesized by relatively conserved pathways. Gibberellins (GAs), which are essential for plant growth and development, are diterpenoid phytohormones. (E,E,E)-Geranylgeranyl diphosphate (GGPP) is the precursor for both GAs and other diterpenoids of secondary metabolism. ent-Kaurene biosynthesis from GGPP is a key step of GA formation, which is catalyzed by two sequential and dedicated diterpene synthases (diTPSs): Ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) of the terpene synthase gene family. Sharing a common evolutionary origin, CPS and KS belong to different TPS subfamilies. Tea plant (Camellia sinensis), the subject of this study, is a leaf-based economic crop. Budbreak mainly manipulated by GAs is a primary factor for targeted tea breeding. The key genes for gibberellin biosynthesis are known; however, they have not yet been characterized in tea plants. Here, we identified and functionally characterized three diterpene biosynthesis-related genes, including one CPS and two highly similar KSs in tea plants. These genes were initially identified through transcriptome sequencing. The functional characterization determined by enzymatic activity assay indicated that CsCPS could catalyze GGPP to form ent-copalyl diphosphate (ent-CPP), which was further used as the substrate by CsKS1 to produce ent-kaurene or by CsKS2 to produce 16α-hydroxy-ent-kaurane with ent-kaurene as a minor product, respectively. We demonstrated that the divergent evolution of diterpene biosynthesis in tea plants resulted from gene duplication of KSs, followed by functional divergence caused by single amino acid variation. This study would provide an insight into the diterpenoid metabolism and GA biosynthesis in tea plants to further understand leaf bud development or insect resistance and to provide a genetic basis for tea plant breeding.