Defining the regulatory role of protein O-fucosylation during pollen-pistil communication

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Plant reproduction is responsible for the generation of new plant embryos with their surrounding starch or oil rich tissues (seeds/fruits) which are used worldwide as important food and energy sources. Additionally, it is becoming increasingly important to reproductively isolate plants growing in a field setting to prevent gene flow between transgenic and non-transgenic variants. The Wallace lab has recently demonstrated that a group of proteins called Protein O-FucosylTransferases (POFTs), which attach sugars onto target proteins to regulate their behavior, play a role in this plant reproduction process. The Wallace lab will use advanced biochemical and genetic methods to dissect the contribution of POFTs in plant reproduction and identify the proteins that are being regulated by these enzymes to elucidate how POFTs contribute to plant reproduction and how these enzymes could be manipulated to optimize this process. Additionally, this project will support the research training of graduate, undergraduate, and high school students from diverse and under-represented backgrounds. This project will also support the development of web-based video protocols that communicate the advanced methods used to approach this research in a freely available manner. The long-term goal is to mechanistically understand the enzymes that catalyze protein O-fucosylation, their protein targets, and the extent of this modification in plant systems. The central hypothesis guiding this research is that POFTs fucosylate target proteins that govern cell wall polysaccharide biosynthesis, cell adhesion, and cell-cell communication in plants. Specific Aim 1 seeks to understand the enzymology and sugar nucleotide substrate specificity of Arabidopsis putative POFTs by employing sugar nucleotide hydrolysis assays to understand which sugar nucleotides are substrates for putative POFTs. Specific Aim 2 will employ biotin proximity labeling and advanced quantitative proteomic analysis to identify putative protein glycosylation substrates of these POFTs. Specific Aim 3 will utilize affinity capture methods and advanced proteomic analysis to broadly identify proteins in the Arabidopsis proteome that are subject to post-translational protein O-fucosylation, thus providing a catalog of proteins that are modified in this manner. The unifying goal of this research is to elucidate the role of protein O-fucosylation in plant systems, with a particular focus on plant sexual reproduction, and to understand how these post-translational modifications actuate diverse processes, such as cell adhesion and cell-cell communication.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.