Protocols for Isolating and Characterizing Polysaccharides From 1 Plant Cell Walls: A Case Study Using Rhamnogalacturonan-II

Background: In plants, there is a large diversity of polysaccharides that comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin modification and valorization has attracted much attention due to its expanding roles of pectin in biomass deconstruction, food science, material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of pectin structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model.Results: We outline how to isolate RG-II from celery and duckweed cell wall material and red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we demonstrate the use of mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG II molecule and RG-II-derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. As RG-II is further modified by non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters, we then describe ways to use electrospray-MS to identify these moieties on RG-II-derived oligosaccharides. We then explored the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) in identifying RG-II-specific sugars and non-glycosyl modifications to complement and extend MS-based approaches. Finally, we describe how to assess the factors that affect RG-35 II dimerization using liquid chromatographic and NMR spectroscopic approaches.Conclusions: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.