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Determining the polysaccharide composition of plant cell walls

Abstract

The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography–mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.

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Figure 1: Strategies for determining the type of analysis most suited for compositional analysis of cell walls and cell wall extracts.
Figure 2: Methods for the conversion of uronic acid residues and their methyl esters in cell wall polysaccharides to their corresponding neutral sugars.
Figure 3: Methylation analysis of cell wall polysaccharides.
Figure 4: Fragmentation of PMAAs during electron ionization MS51.

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Acknowledgements

This work has been supported by grants from the Australian Research Council (ARC Centre of Excellence in Plant Cell Walls), the Grains Research and Development Corporation, the Cooperative Research Centre (CRC) for Industrial Plant Biopolymers, the CRC for Bioproducts, and CSIRO Food Futures over many years. We acknowledge the support of many colleagues, particularly the postdoctoral fellows and research assistants in the Bacic laboratory, who have contributed to the refinement of these methods.

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F.A.P. conducted the experimental and data analysis work. F.A.P., C.W., G.B.F. and A.B. wrote the manuscript.

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Correspondence to Antony Bacic.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

PMAA derivatives. Deduced linkages based on O-acetyl and O-methyl positions, molecular weight (MW) and an example of GC retention time relative to myo-inositol as internal standard (Rt) under the conditions described in the text. (PDF 42 kb)

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Pettolino, F., Walsh, C., Fincher, G. et al. Determining the polysaccharide composition of plant cell walls. Nat Protoc 7, 1590–1607 (2012). https://doi.org/10.1038/nprot.2012.081

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