Single-cell RNA sequencing reveals transcriptional regulation and metabolic pathways of terpenoid biosynthesis in developing Cinnamomum camphora leaf cells

Cinnamomum camphora (Camphor tree) is an economically significant species known for its terpenoid-rich essential oils. However, the molecular mechanisms underlying its leaf development remain poorly understood, especially at the cellular level. In this study, we applied high-throughput single-cell RNA sequencing (scRNA-seq) to profile the transcriptomic landscape of developing C. camphora leaves, identifying eight distinct cell populations, including mesophyll, epidermal, guard, vascular, and proliferating cells. Pseudotime trajectory analysis revealed the dynamic progression of mesophyll cell differentiation, with three distinct developmental states observed as cells transitioned from proliferating to differentiated stages. We identified key metabolic pathways involved in terpenoid biosynthesis, lipid metabolism, and photosynthesis. Notably, genes such as 4CLL9 , CHLP or GPPS1 showed cell-type-specific expression in mesophyll or epidermal cells. Additionally, transcription factors from the MYB and bHLH families were enriched in specific cell types, regulating secondary metabolism and hormone signaling. This study not only provides a detailed transcriptomic atlas of C. camphora leaf development but also uncovers novel cell-type-specific marker genes, key regulatory networks, and metabolic pathways, offering valuable resources for future investigations into terpenoid metabolism and cellular differentiation in woody species. • Single-cell RNA sequencing revealed eight distinct cell types in developing C. camphora leaves, including mesophyll, epidermal, vascular, and guard cells. • Terpenoid biosynthesis in C. camphora is regulated in a cell-type-specific manner, with key genes such as 4CLL9, CHLP , and VTE3 enriched in mesophyll cells, while GGPS1 , ELI3 , and NAAT1 exhibit specificity in epidermis-like and guard mother cells. • Pseudotime analysis revealed three distinct mesophyll developmental states with photosynthesis genes enriched in later stages and hormone signaling genes in early stages. • New marker genes for C. camphora leaf cells were identified, including ND6 for proliferating cells, GSTU25 for mesophyll cells, and Cca.gene39283 for vascular cells, providing valuable resources for future plant scRNA-seq studies.