Single cell mass spectrometry reveals intercellular compartmentalization of camptothecin biosynthesis in the tree Camptotheca acuminata

Abstract

The medicinal tree Camptotheca acuminata produces camptothecin, a monoterpenoid indole al- kaloid (MIA) precursor for several leading chemotherapeutic agents (Lorence and Nessler 2004). Alt- hough the biosynthesis of camptothecin remains poorly understood, a putative route has been hypothe- sized based on in planta metabolite profiling and feeding studies (Fig. 1a) (Sheriha and Rapoport 1976; Sadre et al. 2016). However, pathways proposed on whole-tissue or organ-level metabolomic and tran- scriptomic data lack resolution on the intricate, cell-specific compartmentalization of natural products biosynthesis. Such gaps could be addressed by single cell technologies, which have recently shown tremendous potential to transform gene discovery in herbaceous plants (Li et al. 2023; Zhan et al. 2023; Vu et al. 2024; Wu et al. 2024; McClune et al. 2025). Nevertheless, single cell mass spectrometry (scMS) has not been adapted for woody species, largely due to the challenges associated with their highly lignified tissue and complex cellular architecture. In this study, we developed an scMS pipeline for the woody tree C. acuminata to investigate the intercellular organization of camptothecin biosynthesis. O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/661346v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17f7710org.highwire.dtl.DTLVardef@161c469org.highwire.dtl.DTLVardef@1a5ee1borg.highwire.dtl.DTLVardef@b361b9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Single cell metabolomics of Camptotheca acuminata. (a) Proposed camptothecin biosynthetic pathway. (b) Schematic experimental pipeline. (c) Dot plots showing concentrations of key metabolites in single cells across organs. Each data point rep- resents a protoplast. For each metabolite, dots were offset from the center based on the distribution of concentrations among sin- gle cells. (N.D.: not detected). (d) Stacked bars showing concentrations in individual cells of loganic acid, secologanic acid, strictosidinic acid, strictosamide, pumiloside, and camptothecin). Cells are organized into groups with loganic acid only (A), secologanic acid only (B), loganic and secologanic (C), strictosidinic acid only (D), and other alkaloids (strictosamide, pumiloside, and camptothecin) (E). In each group, cells are ordered by total metabolite concentration with highest concentration in the center and lower producers closer to the edges. Panel widths scale with the number of cells in each group, and colours cor- respond to individual metabolites. C_FIG