EAGER: Comparative single cell transcriptomics and regulomics: A proof-of-concept application of cutting-edge -omics techniques with non-model systems

University Of Georgia Research Foundation Inc, Athens GA

Investigators

Abstract

Plants produce diverse chemicals, known as natural products, that function to deter pests, attract pollinators, and interact with microbes. While recent technologies have enabled the discovery of the enzymes responsible for natural product production, there is limited knowledge of how plants regulate production of these compounds. Emerging studies have shown that a growing number of plant natural products are produced in distinct cell types, suggesting strict regulation of their biosynthesis. This project will focus on the monoterpene indole alkaloids, a class of diverse specialized metabolites, some of which have potent biological and pharmaceutical activities. The goal will be to discover the regulatory components of monoterpene indole alkaloid biosynthesis in Catharanthus roseus, Madagascar periwinkle, and its relative, Camptotheca acuminata. Through this project, data and methods will be developed to elucidate the molecular mechanisms by which genes are regulated. Undergraduates will be trained in state-of-the-art genetic methods, computational analyses, and data analysis methods associated with plant biology. Through this project, new tools for biotechnology and engineering the production of natural products in plants will be created. Accumulating evidence suggests plant specialized metabolism is under exquisite and strict regulation at the organ, tissue, and cell type levels, thereby leading to distinct cell type and subcellular localization of natural products. How the genes responsible for the biosynthesis of natural products are regulated is not well understood. Monoterpene indole alkaloids (MIA) are a class of diverse specialized metabolites produced by plants in the Order Gentianales, including C. roseus and the Asterid species C. acumuinata. In C. roseus, the MIA biosynthetic pathway is sequentially partitioned into three discrete cell types, suggestive of complex regulatory networks controlling the biosynthesis and transport of intermediates. In other species, however, the localization and cell type specificity of MIA remain an enigma. While the early steps of the MIA biosynthetic pathway are conserved among C. roseus and C. acuminata, the downstream stages are divergent. The knowledge of two related MIA biosynthetic pathways at the gene, cell type, and regulatory levels will shed light on the regulation and evolution of these complex natural products, as well as how the pathway can be reconstructed and modified in heterologous systems. This project will demonstrate the viability of single cell transcriptomics and chromatin accessibility profiling for C. acuminata to understand the cell type specificity of MIA biosynthetic genes across leaf cells and the extent of conservation between C. roseus and C. acuminata. This project will also demonstrate the viability of DNA affinity sequencing (DAP-seq) in C. roseus and C. acuminata as a method of bridging transcription factors and cis-regulatory elements identified by computational methods. Taken together, this project will establish best practices for two cutting edge genomics technologies for plants and potentially deepen our understanding of the regulatory landscapes of plant specialized metabolism. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.