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Determining the polysaccharide composition of plant cell walls

Nature Protocols volume 7, pages 1590–1607 (2012)Cite this article

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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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References

  1. Bacic, A., Harris, P.J. & Stone, B.A. Structure and function of plant cell walls. in The Biochemistry of Plants (ed. Priess, J.) 297–371 (Academic Press, 1988).

  2. Carpita, N.C. & Gibeaut, D.M. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1–30 (1993).

    PubMed  CAS  Google Scholar 

  3. Jacobs, D.R. & Gallaher, D.D. Whole grain intake and cardiovascular disease: a review. Curr. Atheroscler. Rep. 6, 415–423 (2004).

    PubMed  Google Scholar 

  4. Jemal, A., Ward, E., Hao, Y.P. & Thun, M. Trends in the leading causes of death in the United States, 1970–2002. JAMA 294, 1255–1259 (2005).

    PubMed  CAS  Google Scholar 

  5. Dikeman, C.L. & Fahey, G.C. Viscosity as related to dietary fiber: a review. Crit. Rev. Food Sci. Nutr. 46, 649–663 (2006).

    PubMed  CAS  Google Scholar 

  6. Collins, H.M. et al. Variability in the fine structures of non-cellulosic cell wall polysaccharides from cereal grains: potential importance in human health and nutrition. Cereal Chem. 87, 272–282 (2010).

    CAS  Google Scholar 

  7. Mohnen, D. Pectin structure and biosynthesis. Curr. Opin. Plant Biol. 11, 266–277 (2008).

    PubMed  CAS  Google Scholar 

  8. Doblin, M.S., Pettolino, F.A. & Bacic, A. Plant cell walls: the skeleton of the plant world. Funct. Plant Biol. 37, 357–381 (2010).

    CAS  Google Scholar 

  9. Burton, R.A., Gidley, M.J. & Fincher, G.B. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat. Chem. Biol. 6, 724–732 (2010).

    PubMed  CAS  Google Scholar 

  10. Moller, I. et al. High throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J. 50, 1118–1128 (2007).

    PubMed  CAS  Google Scholar 

  11. Fry, S.C., Nesselrode, B.H.W.A., Miller, J.G. & Mewburn, B.R. Mixed-linkage (1,3;1,4)-β-D-glucan is a major hemicellulose of Equisetum (horsetail) cell walls. New Phytol. 179, 104–115 (2008).

    PubMed  CAS  Google Scholar 

  12. Sørensen, I. et al. Mixed-linkage (1→3), (1→4)-β-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant Journal 54, 510–521 (2008).

    Google Scholar 

  13. Mansfield, S.D., Kim, H., Lu, F. & Ralph, J. Whole plant cell wall characterization using 2D-NMR. Nat. Protoc. 7, 1579–1589 (2012).

    PubMed  CAS  Google Scholar 

  14. Willför, S. et al. Carbohydrate analysis of plant materials with uronic acid-containing polysaccharides–a comparison between different hydrolysis and subsequent chromatographic analytical techniques. Ind. Crops Prod. 29, 571–580 (2009).

    Google Scholar 

  15. Obel, N. et al. Microanalysis of plant cell wall polysaccharides. Mol. Plant 2, 922–932 (2009).

    PubMed  CAS  Google Scholar 

  16. Goubet, F., Jackson, P., Deery, M.J. & Dupree, P. Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal. Biochem. 300, 53–68 (2002).

    PubMed  CAS  Google Scholar 

  17. McCann, M.C. et al. Approaches to understanding the functional architecture of the plant cell wall. Phytochemistry 57, 811–821 (2001).

    PubMed  CAS  Google Scholar 

  18. Wilson, S.M. & Bacic, A. Preparation of plant tissue sections for electron microscopy. Nat. Protoc. 7, in press (2012).

  19. Knox, J.P. Revealing the structural and functional diversity of plant cell walls. Curr. Opin. Plant Biol. 11, 308–313 (2008).

    PubMed  CAS  Google Scholar 

  20. Fry, S.C. The Growing Plant Cell Wall: Chemical and Metabolic Analysis (Longman Scientific & Technical, 1988).

  21. Fincher, G.B. Morphology and chemical composition of barley endosperm cell walls. J. Inst. Brew. 81, 116–122 (1975).

    CAS  Google Scholar 

  22. Galambos, J.T. The reaction of carbazole with carbohydrates. I. Effect of borate and sulfamate on the carbazole colour of sugars. Anal. Biochem. 19, 119–132 (1967).

    PubMed  CAS  Google Scholar 

  23. Blumenkrantz, N. & Asboe-Hansen, G. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489 (1973).

    PubMed  CAS  Google Scholar 

  24. Filisetti-Cozzi, T.M.C.C. & Carpita, N.C. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197, 157–162 (1991).

    PubMed  CAS  Google Scholar 

  25. Oxley, D., Currie, G. & Bacic, A. Analysis of carbohydrate from glycoproteins. In Purifying Proteins for Proteomics. A Laboratory Manual (ed. Simpson, R.J.) pp 579–637 (Cold Spring Harbor Laboratory Press, 2004).

  26. Kim, J.B. & Carpita, N.C. Changes in esterification of the uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiol. 98, 646–653 (1992).

    PubMed  PubMed Central  CAS  Google Scholar 

  27. Albersheim, P., Nevins, D.J., English, P.D. & Karr, A. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5, 340–345 (1967).

    CAS  Google Scholar 

  28. Blakeney, A.D., Harris, P.J., Henry, R.J. & Stone, B.A. A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr. Res. 113, 291–299 (1983).

    CAS  Google Scholar 

  29. Saeman, J.F., Moore, W.E. & Millet, M.A. Sugar units present. Hydrolysis and quantitative paper chromatography. In Methods in Carbohydrate Chemistry (ed. Whistler, R.L.) pp 54–69 (Academic Press, 1983).

  30. Stevenson, T.T. & Furneaux, R.H. Chemical methods for the analysis of sulphated galactans from red algae. Carbohydr. Res. 210, 277–298 (1991).

    PubMed  CAS  Google Scholar 

  31. York, W.S., Darvill, A.G., McNeil, M. & Albersheim, P. 3-Deoxy-d-manno-2-octulosonic acid (KDO) is a component of Rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants. Carbohydr. Res. 138, 109–126 (1985).

    CAS  Google Scholar 

  32. Doares, S.H., Albersheim, P. & Darvill, A.G. An improved method for the preparation of standards for glycosyl-linkage analysis of complex carbohydrates. Carbohydr. Res. 210, 311–317 (1991).

    CAS  Google Scholar 

  33. Ciucanu, I. Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry. Anal. Chim. Acta 576, 147–155 (2006).

    PubMed  CAS  Google Scholar 

  34. Hakomori, S.-I. A rapid permethylation of glycolipid, and polysaccharide catalysed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem. 55, 205–208 (1964).

    PubMed  CAS  Google Scholar 

  35. Ciucanu, I. & Kerek, F. A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131, 209–217 (1984).

    CAS  Google Scholar 

  36. Anumula, K.R. & Taylor, P.B. A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203, 101–108 (1992).

    PubMed  CAS  Google Scholar 

  37. Laine, C., Tamminen, T., Vikkula, A. & Vuorinen, T. Methylation analysis as a tool for structural analysis of wood polysaccharides. Holzforschung 56, 607–614 (2002).

    CAS  Google Scholar 

  38. Sims, I.M., Munro, S.L.A., Currie, G., Craik, D. & Bacic, A. Structural characterisation of xyloglucan secreted by suspension-cultured cells of Nicotiana plumbaginifolia. Carbohydr. Res. 293, 147–172 (1996).

    PubMed  CAS  Google Scholar 

  39. Kim, J.S., Reuhs, B.L., Michon, F., Kaiser, R.E. & Arumugham, R.G. Addition of glycerol for improved methylation linkage analysis of polysaccharides. Carbohydr. Res. 341, 1061–1064 (2006).

    PubMed  CAS  Google Scholar 

  40. Pettolino, F. et al. Hyphal cell walls from the plant pathogen Rhynchosporium secalis contain (1,3/1,6)-β-D-glucans, galacto- and rhamnomannans, (1,3;1,4)-β-D-glucans and chitin. FEBS J. 276, 4122–4133 (2009).

    Google Scholar 

  41. Needs, P.W. & Selvendran, R.R. A critical assessment of a one-tube procedure for the linkage analysis of polysaccharides as partially methylated alditol acetates. Carbohydr. Res. 254, 229–244 (1994).

    CAS  Google Scholar 

  42. Lau, E. & Bacic, A. Capillary gas chromatography of partially methylated alditol acetates on a high-polarity, cross-linked, fused silica BPX70 column. J. Chrom. 637, 100–103 (1993).

    CAS  Google Scholar 

  43. Shea, E.M., Gibeaut, D.M. & Carpita, N.C. Structural analysis of the cell walls generated by carrot protoplasts. Planta 179, 293–308 (1989).

    PubMed  CAS  Google Scholar 

  44. Sims, I.M. & Bacic, A. Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia. Phytochemistry 38, 1397–1405 (1995).

    CAS  Google Scholar 

  45. Zhu, Y., Pettolino, F., Mau, S.-L. & Bacic, A. Characterization of cell wall polysaccharides from the medicinal plant Panax notoginseng. Phytochemistry 66, 1067–1076 (2005).

    PubMed  CAS  Google Scholar 

  46. Lewis, D., Bacic, A., Chandler, P.M. & Newbigin, E.J. Aberrant cell expansion in the elongation mutants of barley. Plant Cell Physiol. 50, 554–571 (2009).

    PubMed  CAS  Google Scholar 

  47. Zhang, Q. et al. Cell wall modifications in maize pulvini in response to gravitational stress. Plant Physiol. 156, 2155–2171 (2011).

    PubMed  PubMed Central  CAS  Google Scholar 

  48. Prabasari, I., Pettolino, F., Liao, M.-L. & Bacic, A. Pectic polysaccharides from mature orange (Citrus sinensis) fruit albedo cell walls: sequential extraction and chemical characterisation. Carbohydr. Polym. 84, 484–494 (2011).

    CAS  Google Scholar 

  49. Updegraff, D.M. Semimicro determination of cellulose in biological materials. Anal. Biochem. 32, 420–424 (1969).

    PubMed  CAS  Google Scholar 

  50. Zablackis, E., Huang, J., Muller, B., Darvill, A.G. & Albersheim, P. Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves. Plant Physiol. 107, 1129–1138 (1995).

    PubMed  PubMed Central  CAS  Google Scholar 

  51. Björndal, H., Hellerqvist, C.G., Lindberg, B. & Svensson, S. Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides. Angew. Chem. Int. Ed. 9, 610–619 (1970).

    Google Scholar 

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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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Authors and Affiliations

  1. Commonwealth Scientific and Industrial Research Organization (CSIRO) Plant Industry, Canberra, Australia

    Filomena A Pettolino

  2. Australian Research Council (ARC) Centre for Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Melbourne, Australia

    Cherie Walsh & Antony Bacic

  3. ARC Centre for Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, Australia

    Geoffrey B Fincher

Authors
  1. Filomena A Pettolino
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  2. Cherie Walsh
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  4. Antony Bacic
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Contributions

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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