An international team of researchers from the University of Georgia and the Max Planck Institute (MPI) for Chemical Ecology in Germany have discovered a promising strategy to decode the metabolic pathways for plant compounds important in medical treatments, according to a new study published in Nature Chemical Biology.
Led by Chenxin Li of UGA's Center for Applied Genetic Technologies in the College of Agricultural and Environmental Sciences, the research team studied the biosynthesis of two alkaloids from the Madagascar periwinkle (Catharanthus roseus) that are used as anti-cancer agents in human medicine. The genes for the formation of these active substances are expressed in different cell types. By using single-cell analyses, the scientists were able to discover new genes important for biosynthesis and show that the intermediates of the metabolic pathway accumulate in specific cell types. Intermediates are molecules that are the precursors or metabolites of biologically significant molecules.
The researchers predict that this methodological approach will provide important new insights into the biosynthesis of many other natural products from the plant kingdom.
Plants are impressive in their diversity, especially in the variety of metabolites they produce. Many natural plant products are composed of highly complex molecules, such as the alkaloids vincristine and vinblastine, which are produced by the Madagascar periwinkle. These two substances are already indispensable in cancer therapy, and researchers are interested in finding out which individual biosynthetic steps are required to form the complex molecules.
“Currently, these compounds are still obtained in very small quantities from the plant's leaf extract. We can learn from the plant how this compound is produced and use this knowledge to develop production systems that are more cost-effective, scalable and sustainable,” said Li, first author of the study, describing the research goal.
Investigating specialized cell types
Scientists know that gene activity differs among the cells of a plant and that the chemistry can differ drastically from cell to cell. The goal of the recent study was to use a new set of methods collectively termed single-cell omics to investigate specialized and rare cell types that play a central role in the biosynthesis of plant natural products, and whose signals are often obscured by more abundant cell types in plant organs.
“With single-cell omics, we have a method that allows researchers to assign genetic and metabolic information to individual cells. The term ‘omics’ refers to the fact that an entire collection of genes or metabolites is quantified and analyzed,” explained Lorenzo Caputi, head of the Alkaloid Biosynthesis Project Group in the Department of Natural Product Biosynthesis at MPI and one of the lead authors.
Understanding biosynthetic pathways
As the analyses showed, the entire biosynthetic pathway for the alkaloid vinblastine is organized in three stages and three discrete cell types.
"The first stage is expressed exclusively in specialized cells associated with vascular bundles in the leaf, called IPAP. The second stage of the biosynthetic pathway is expressed only in cells of the epidermis, the layer of cells that cover the leaves, and the last known steps of the biosynthetic pathway are expressed exclusively in idioblasts, a rare cell type of the leaf," Li summarized.
The researchers measured the concentrations of several intermediates in the metabolic pathway for vinblastine in single cells and were surprised by the results.
"Two important precursors of vinblastine — catharanthine and vindoline — occur in the idioblast cells at millimolar concentrations, about three orders of magnitude higher than vinblastine itself. The concentration of the two precursors in these cells was much higher than we expected and even exceeded their concentrations in whole organ extracts. However, this observation makes sense in that catharanthine and vindoline were found only in the rare idioblast cells. The abundant other cells in the leaf dilute the high concentration when whole leaves are crushed," said Sarah O’Connor, head of the Department of Natural Product Biosynthesis at MPI.
New techniques to study natural products
The research team is confident that the organization of biosynthetic pathways for medicinally relevant alkaloids in Catharanthus roseus is not an isolated phenomenon. "We are just beginning to understand how and why such a cell type-specific organization exists. In addition, analysis of genes expressed simultaneously in a particular cell type has helped us identify new players in this metabolic pathway, said Robin Buell, the Georgia Research Alliance (GRA) Eminent Scholar Chair in Crop Genomics at UGA. “The same technique can be used to study the biosynthesis of many other natural products. Finally, the exact sites of accumulation of plant compounds, such as the epidermis, the vascular system or latex duct, can help us hypothesize the ecological roles of natural products. For example, depending on the pattern of accumulation, the compounds may be more effective against biting insects than they are against sap-sucking insects."
A better understanding of the biosynthetic pathways of the anti-cancer drugs vincristine and vinblastine may also help to produce or harvest these compounds more effectively in the long term. The use of methods described is also promising for the study of many other interesting and medically important natural products from the plant kingdom.
The approach described in the study will help to narrow down these rare and specialized cells and uncover the gene activities and chemistry that are exclusive to them.