Cell-Specific Expression and Cellular Compartmental Regulation in Camptothecin Biosynthesis.

Plant secondary metabolites such as monoterpenoid indole alkaloids (MIAs) show tightly regulated biosynthesis and accumulation in specific organelles of distinct cell types. However, the cell-specific expression patterns of anticancer MIA camptothecin biosynthetic genes in Ophiorrhiza pumila and the regulatory mechanisms of camptothecin biosynthesis via cellular compartmentalisation remain poorly understood. Here, single-cell RNA sequencing of O. pumila leaves generated 9181 cells, classified into four types using marker gene annotation and in situ hybridisation. Gene expression profiling combined with pseudotime analyses revealed that camptothecin biosynthetic genes are preferentially expressed in epidermal and young cells. Notably, the genes encoding camptothecin key enzymes, strictosidine synthase (STR) and tryptophan decarboxylase (TDC), have expanded within the O. pumila STR-TDC gene cluster, supporting cell-specific specialisation. Concurrently, this gene cluster has evolved a novel functional gene, OpAVT1, which annotated as a vacuolar transporter, mediates the transport of tryptophan from vacuole to the cytosol, thereby regulating camptothecin and other MIAs accumulation. Collectively, these findings establish a cellular-level framework for camptothecin biosynthesis and regulation.