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Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE)

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Abstract

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear β(1,4) galactan and linear α(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.

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Abbreviations

AMAC:

2-Aminoacridone

ANTS:

8-Aminonaphthalene-1,3,6 trisulphonic acid

Ara:

Arabinose

CWM:

Cell wall material

DM:

Degree of methylation

DP:

Degree of polymerisation

endo-PG:

Endopolygalacturonase

FTIR:

Fourier transform infra-red spectroscopy

Gal:

Galactose

GalU:

Galacturonic acid

PACE:

Polysaccharide analysis by carbohydrate gel electrophoresis

PGA:

Polygalacturonan

MS:

Mass spectrometry

OGA:

Oligogalacturonan

RGI:

Rhamnogalacturonan I

RGII:

Rhamnogalacturonan II

WT:

Wild type

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Acknowledgements

We thank Julie Howard and Kathryn Lilley (Cambridge Centre for Proteomics, University of Cambridge) for their help on mass spectrometric analyses, and Tom Dunkley for critical reading of the manuscript. We thank Syngene (Cambridge, UK) for the gift of GeneTools software. We thank Yoichi Tsumuraya (Saitama, Japan) for the gift of galacto-oligosaccharides. We thank Elner Rathbone, (Sigma, UK) for providing some reagents for MALDI-TOF MS. The work was funded by grants from the BBSRC and a BBSRC studentship to CJB.

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Author notes

  1. Louise E. Tailford

    Present address: Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, The Medical School, NE2 4HH, Newcastle upon Tyne, UK

  2. Helen Welchman

    Present address: Drug Control Centre, King’s College London, 150 Stamford Street, SE19NH, London, UK

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Building O, Downing Site, CB2 1QW, Cambridge, UK

    Christopher J. Barton, Louise E. Tailford, Helen Welchman, Zhinong Zhang, Paul Dupree & Florence Goubet

  2. Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, The Medical School, NE2 4HH, Newcastle upon Tyne, UK

    Harry J. Gilbert

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  1. Christopher J. Barton
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Correspondence to Paul Dupree.

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Barton, C.J., Tailford, L.E., Welchman, H. et al. Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Planta 224, 163–174 (2006). https://doi.org/10.1007/s00425-005-0185-9

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  • DOI: https://doi.org/10.1007/s00425-005-0185-9

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