Understanding plant natural product biosynthesis using single-cell mass spectrometry

Plants produce a large array of valuable natural products. These compounds are synthesized through the sequential action of dedicated enzymes, which can be located within different cell compartments or sometimes in different cell types. Therefore, elucidation of the biosynthesis of plant natural products would be greatly facilitated by mapping the location of metabolic intermediates and final products at the single-cell resolution. However, detection, identification, and quantification of metabolites in single cells, particularly from plants, have remained a significant challenge. In this thesis, a robust plate-based single-cell mass spectrometry method (scMS) is developed, enabling the identification and quantification of 16 different metabolites across four chemical classes of natural products in individual cells from leaves, roots, and petals of Catharanthus roseus, a medicinal plant well-known for producing the anticancer drug vinblastine. The scMS data reveal the substantial heterogeneity of metabolite accumulation among cell populations, and show that cell-specific metabolite localization patterns vary among organs. This scMS method is then combined with stable isotope labeling, allowing real-time tracking of plant natural product synthesis and transport at the single-cell level. Furthermore, single-cell RNA-sequencing is also integrated with the scMS approach, enabling simultaneous measurement of both gene expression and metabolite levels from the same individual plant cell. Overall, the single-cell MS-based methods developed in this thesis provide new mechanistic insights into the synthesis, trafficking, and storage of plant natural products at the cellular level.