Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II
Abstract
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-derivedmore »
- Publication Date:
- Research Org.:
- Univ. of Georgia, Athens, GA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; National Institutes of Health (NIH)
- OSTI Identifier:
- 1798000
- Alternate Identifier(s):
- OSTI ID: 1851080
- Grant/Contract Number:
- DESC0008472; DESC0015662; SC0008472; SC0015662; 1S10OD018530
- Resource Type:
- Published Article
- Journal Name:
- Biotechnology for Biofuels
- Additional Journal Information:
- Journal Name: Biotechnology for Biofuels Journal Volume: 14 Journal Issue: 1; Journal ID: ISSN 1754-6834
- Publisher:
- Springer Science + Business Media
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; Biotechnology & Applied Microbiology; Energy & Fuels; Rhamnogalacturonan-II (RG-II); Pectin; Plant cell wall; Borate diester; SEC-MALS; NMR spectroscopy
Citation Formats
Barnes, William J., Koj, Sabina, Black, Ian M., Archer-Hartmann, Stephanie A., Azadi, Parastoo, Urbanowicz, Breeanna R., Peña, Maria J., and O’Neill, Malcolm A. Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. Netherlands: N. p., 2021.
Web. doi:10.1186/s13068-021-01992-0.
Barnes, William J., Koj, Sabina, Black, Ian M., Archer-Hartmann, Stephanie A., Azadi, Parastoo, Urbanowicz, Breeanna R., Peña, Maria J., & O’Neill, Malcolm A. Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. Netherlands. https://doi.org/10.1186/s13068-021-01992-0
Barnes, William J., Koj, Sabina, Black, Ian M., Archer-Hartmann, Stephanie A., Azadi, Parastoo, Urbanowicz, Breeanna R., Peña, Maria J., and O’Neill, Malcolm A. Tue .
"Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II". Netherlands. https://doi.org/10.1186/s13068-021-01992-0.
@article{osti_1798000,
title = {Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II},
author = {Barnes, William J. and Koj, Sabina and Black, Ian M. and Archer-Hartmann, Stephanie A. and Azadi, Parastoo and Urbanowicz, Breeanna R. and Peña, Maria J. and O’Neill, Malcolm A.},
abstractNote = {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.},
doi = {10.1186/s13068-021-01992-0},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 14,
place = {Netherlands},
year = {Tue Jun 22 00:00:00 EDT 2021},
month = {Tue Jun 22 00:00:00 EDT 2021}
}
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