Cloning and Functional Characterization of PmCPS, a Copalyl Diphosphate Synthase Gene from Pinus massoniana Lamb

Pinus massoniana Lamb. is an important timber- and oleoresin-producing tree species in southern China. Copalyl diphosphate synthase (CPS) catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP), representing a key step in the biosynthesis of gibberellins (GAs) and diverse diterpenoids. In this study, PmCPS was cloned from P. massoniana, and its protein structure, phylogenetic relationships, tissue-specific expression pattern, and promoter cis-acting elements were characterized. Its biological function was further investigated using virus-induced gene silencing (VIGS) in P. massoniana and heterologous overexpression in Arabidopsis thaliana. PmCPS exhibited the characteristic structural features of plant diterpene synthases. PmCPS transcripts were most abundant in needles, reaching approximately 90 times the level in female strobili, and their abundance increased significantly to about 1.7 times the control level 3 h after GA3 treatment. Silencing PmCPS reduced CPS enzymatic activity from approximately 16.8 to 12.0 IU g−1 and decreased GA3 content from approximately 57 to 46 ng g−1, and downregulated genes associated with the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and GA biosynthesis. Conversely, heterologous expression of PmCPS increased CPS activity to 1.60–1.98 times the wild-type level and endogenous GA3 content from 43.90 to 71.50–80.39 ng g−1 in A. thaliana, accumulated more than twice the fresh weight of wild-type plants, and significantly accelerated bolting. Under abscisic acid (ABA), methyl jasmonate (MeJA), NaCl, and D-mannitol treatments, the transgenic plants maintained greater primary root growth than wild-type plants. Collectively, these findings indicate that PmCPS is a key component of GA-related diterpenoid metabolism in P. massoniana and may contribute to plant growth, development, and treatment responses by modulating plastidial isoprenoid metabolism and phytohormone homeostasis. This study advances our understanding of diterpenoid metabolism and hormonal regulation in P. massoniana and identifies PmCPS as a promising candidate gene for molecular breeding targeting growth- and stress-related traits.