EAGER: Synthesis and Implementation of Click Sugar Analogs for Plant Cell Wall Polysaccharide Imaging

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Nontechnical:All plant cells are surrounded by a polysaccharide-rich cell wall comprised of cellulose, hemicellulose, and pectin. While plant cell wall polysaccharides collectively represent the most abundant molecules on the planet, many aspects of cell wall formation remain unclear. For example, hemicelluloses and pectins are synthesized within the cell, transported to the plasma membrane, and organized to form a functional cell wall. However, the mechanisms of polysaccharide transport and organization are unknown, primarily due to the fact that these polysaccharides cannot be visualized by traditional methods. The Wallace lab has previously demonstrated that a metabolically incorporated monosaccharide analog compatible with "click" chemistry can be used to circumvent these issues and fluorescently label cell wall polysaccharides. This research supported through the NSF-EAGER mechanism will build upon these findings by developing novel probes to visualize plant cell wall polysaccharides as they move throughout the cell and into the cell wall. By developing techniques to "see" cell wall polysaccharides in their native environment, these tools will facilitate the investigation of plant cell wall polysaccharide synthesis and transport in fundamentally unique ways that are unattainable with existing technologies. The results of this project will provide a deeper understanding of plant cell wall formation, which could be utilized to produce biomass crops with tailored cell wall compositions for human utilization. Technical:Plants annually assimilate 1011 metric tons of CO2, and a large portion of the resulting reduced carbon is partitioned into plant cell walls, the polysaccharide-rich extracellular matrices surrounding all plant cells. Plant cell wall polysaccharides can be grouped into 3 categories: cellulose, hemicellulose, and pectin. While cellulose is synthesized at the plasma membrane, hemicelluloses and pectins are synthesized in the Golgi apparatus, delivered to the plasma membrane via a poorly characterized vesicle-mediated trafficking pathway, and deposited into the extracellular space, where they become organized to form a functional cell wall. The mechanistic details of non-cellulosic polysaccharide trafficking and organization remain unclear, primarily due to the lack of available methods to visualize plant cell wall polysaccharides. The Wallace group previously demonstrated that Fucose alkyne (FucAl), an analog of the naturally occurring cell wall monosaccharide fucose, is metabolically incorporated into plant tissue and can be fluorescently labeled with azide-containing fluorophores using the copper-catalyzed azide-alkyne cycloaddition reaction. This sugar analog specifically reports the sites of fucose incorporation into pectin and can be used to examine pectin dynamics in native cell walls. This project will build on these ideas by synthesizing and implementing novel monosaccharide analogs of common cell wall sugars that are compatible with click chemistry, and utilizing these metabolic probes to label unique cell wall polysaccharides. Rationally-designed azide-containing monosaccharide analogs will be synthesized and will be assayed for incorporation into cell walls. Furthermore, the labeled polysaccharides, incorporation pathway metabolic enzymes, and reactivity of individual sugars will be investigated. This project will fundamentally change the way that cell wall polysaccharide trafficking is investigated, and will provide key insights into the nanoscale structure and trafficking of non-cellulosic cell wall polysaccharides. Additionally, this project will support advanced graduate education in the developing field of plant chemical biology, will provide advanced undergraduate research education through the UNR Department of Biochemistry senior thesis program, and will support an inter-departmental chemical ecology and chemical biology colloquium.