This DOE Center renewal proposal will focus on structure, biosynthesis, and function of the plant cell wall polysaccharide rhamnogalacturonan-I (RG-I), a major ubiquitous and structurally complicated pectic polysaccharide that exists in the cell walls of all vascular plants. Plant biomass is an abundant renewable resource for the production of carbon-based bioproducts, chemicals, and glycomaterials. However, due to its complexity, there are still major gaps in our understanding of the structural and molecular organization of diverse plant cell walls. Development of new analytical technologies to elucidate the structure, synthesis, and roles of RG-I in wall architecture will provide critical insights into how carbon-rich cell walls are assembled and modified. In the next funding cycle, we propose a set of complementary strategies to elucidate the structure of RG-I that address several factors that have presented challenges to the analysis of RG-I. We have developed a library of carbohydrate active enzymes for deconstructing specific linkages within cell wall polysaccharides. These enzymes will be used to obtain RG-I fragments that can be structurally characterized with spectrometric methods. A new approach that we are developing, MALDI-TOF MS fingerprinting, will allow for high-throughput screening of oligosaccharides extracted from different species and tissue types. We will also pursue both a top-down and bottom-up approach to the analysis of RG-I Chemical Structure. Cell wall matrices are made up of insoluble components that resist spectroscopic analysis. We will continue to optimize the use of ionic liquids and permethylation enabling unbiased carbohydrate analysis of intact biomass and improving the recovery of galacturonic acid and neutral sugars for analysis. We will use new advanced MS/MS and permethylated NMR methods to analyze larger RG-I fragments derived from both enzymatic synthesis and natural sources to elucidate the backbone structures and other glycoconjugates attached to the RG-I. To further our understanding of RGI biosynthesis and function, we will use the robust HEK293 cell heterologous protein expression system developed at the CCRC and previously used by us to express functional plant cell wall glycosyltransferases (GTs) to study the mechanism of RG-I backbone elongation, and the identity and characteristics of enzymes that add the glycosyl and nonglycosyl substituents onto the backbone. With the discovery of several RG-I biosynthetic activities including RG-I backbone GTs and acetyltransferases, target genes have been selected to study the biological function in model plant species. The focused technologies developed through our RGI research will address key challenges in the complex carbohydrate field. By applying these tools to understand the structure, function, and biosynthesis of difficult-to-characterize polysaccharides and glycoproteins, we will continue our expansive collaboration efforts with researchers outside our field, leading to high-quality publications. The DOE center will ensure that these techniques are disseminated with the broader scientific community through annual workforce training and workshops. Additionally, CCRC faculty and staff will mentor graduate students and post-docs in the RGI research program. Through extensive outreach and collaboration, these cutting-edge technologies will be accessible to the scientific community.
Funder: U.S. Department of Energy
Amount: $2,805,000
PI: Parastoo Azadi, Complex Carbohydrate Research Center