Single cell omics extends metabolic regulon via orthologous transcription factors from a pair of medicinal plant species

The medicinal plant Camptotheca acuminata is a source of camptothecin, the starting material for several chemotherapeutic agents. Camptothecin is derived from the monoterpene indole alkaloid (MIA) strictosidinic acid, yet most biosynthetic enzymes downstream of strictosidinic acid are unknown. Catharanthus roseus is a medicinal plant that produces MIAs such as the anti-cancer agent vinblastine currently in use in the clinic. MIA biosynthesis in C. roseus is cell type specific, with the final stage of vinblastine biosynthesis restricted to the rare cell type idioblast. However, the cell type specificity of camptothecin biosynthesis is unknown. Through single cell multi-omics experiments, we detected rare MIA biosynthetic cells in C. acuminata leaf and stem. Metabolite feeding and single cell metabolomics identified stem as the active site for camptothecin biosynthesis. While early intermediates were detected in most cells, late-stage biosynthetic intermediates were detected specifically in a small number of cells within the stem, suggesting strict compartmentalization of the camptothecin biosynthetic pathway. We discovered MYB and bHLH transcription factors (TFs) co-expressed in the same cell type as biosynthetic genes in C. acuminata stem. Interestingly, the C. roseus orthologs of these TFs are idioblast specific and activated MIA biosynthetic genes in C. roseus upon overexpression. Integrating multi-omics and phylogenetic analyses, we generated an extended gene regulatory network for the idioblast metabolic regulon in C. roseus. This study demonstrates that the same clades of TFs have been co-opted to regulate cell type specific MIA biosynthesis across different species. Investigating cell type specific TFs that are co-expressed with biosynthetic genes across multiple species is a powerful strategy to increase the power for discovering plant metabolic regulators.