Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Barnes, William J.; Koj, Sabina; Black, Ian M.; Archer-Hartmann, Stephanie A.; Azadi, Parastoo; Urbanowicz, Breeanna R.; Peña, Maria J.; O’Neill, Malcolm A.

doi:10.1186/s13068-021-01992-0

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Journal Article · Tue Jun 22 00:00:00 EDT 2021 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-021-01992-0· OSTI ID:1798000

Barnes, William J.; Koj, Sabina; Black, Ian M.; Archer-Hartmann, Stephanie A.; Azadi, Parastoo; ; Peña, Maria J.; O’Neill, Malcolm A.

Abstract Background

In plants, a large diversity of polysaccharides 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 valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its 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 walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Ara p ) residue of side chain B is substituted at O- 2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography.

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.

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Research Organization:: Univ. of Georgia, Athens, GA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division

Grant/Contract Number:: SC0008472; SC0015662

OSTI ID:: 1798000

Alternate ID(s):: OSTI ID: 1851080

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Journal Issue: 1 Vol. 14; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

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