, Volume 224, Issue 1, pp 163–174

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

  • Christopher J. Barton
  • Louise E. Tailford
  • Helen Welchman
  • Zhinong Zhang
  • Harry J. Gilbert
  • Paul Dupree
  • Florence Goubet
Original Article


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.


Arabinan Galactan Hydrolase Methylesterified pectin Polygalacturonan Rhamnogalacturonan 





8-Aminonaphthalene-1,3,6 trisulphonic acid




Cell wall material


Degree of methylation


Degree of polymerisation




Fourier transform infra-red spectroscopy




Galacturonic acid


Polysaccharide analysis by carbohydrate gel electrophoresis




Mass spectrometry




Rhamnogalacturonan I


Rhamnogalacturonan II


Wild type


  1. Braithwaite KL, Barna T, Spurway TD, Charnock SJ, Black GW, Hughes N, Lakey JH, Virden R, Hazlewood GP, Henrissat B, Gilbert HJ (1997) Evidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues. Biochemistry 36:15489–15500CrossRefPubMedGoogle Scholar
  2. Burget EG, Reiter WD (1999) The mur4 mutant of Arabidopsis is partially defective in the de novo synthesis of uridine diphospho l-arabinose. Plant Physiol 121:383–389CrossRefPubMedGoogle Scholar
  3. Burget EG, Verma R, Molhoj M, Reiter WD (2003) The biosynthesis of l-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-d-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis. Plant Cell 15:523–531CrossRefPubMedGoogle Scholar
  4. Chen L, Carpita N, Reiter WD, Wilson RH, Jeffries C, McCann MC (1998) A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra. Plant J 16:385–392CrossRefPubMedGoogle Scholar
  5. Gardner SL, Burrell MM, Fry SC (2002) Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides. Phytochemistry 60:241–254CrossRefPubMedGoogle Scholar
  6. Goubet F, Jackson P, Deery M, Dupree P (2002) Polysaccharide analysis using carbohydrate gel electrophoresis (PACE): a method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal Biochem 300:53–68CrossRefPubMedGoogle Scholar
  7. Goubet F, Morriswood B, Dupree P (2003) Analysis of methylated and unmethylated polygalacturonic acid structure by polysaccharide analysis using carbohydrate gel electrophoresis. Anal Biochem 321:174–182CrossRefPubMedGoogle Scholar
  8. Goubet F, Ström A, Dupree P, Williams MAK (2005) An investigation of pectin methylation patterns by two independent techniques: capillary electrophoresis and polysaccharide analysis using carbohydrate gel electrophoresis. Carbohydr Res 340:1193–1199CrossRefPubMedGoogle Scholar
  9. Handford MG, Baldwin TC, Goubet F, Prime TA, Miles J, Yu X, Dupree P (2003) Localisation and characterisation of cell wall mannan polysaccharides in Arabidopsis thaliana. Planta 218:27–36CrossRefPubMedGoogle Scholar
  10. Henrissat B, Coutinho PM, Davies GJ (2001) A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana. Plant Mol Biol 47:55–72CrossRefPubMedGoogle Scholar
  11. Jones L, Milne JL, Ashford D, McQueen-Mason SJ (2003) Cell wall arabinan is essential for guard cell function. Proc Natl Acad Sci USA 100:11783–11788CrossRefPubMedGoogle Scholar
  12. Knox JP (1997) The use of antibodies to study the architecture and developmental regulation of plant cell walls. Int Rev Cytol 171:79–120PubMedGoogle Scholar
  13. Lerouxel O, Choo TS, Seveno M, Usadel B, Faye L, Lerouge P, Pauly M (2002) Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting. Plant Physiol 130:1754–1763CrossRefPubMedGoogle Scholar
  14. Limberg G, Korner R, Buchholt HC, Christensen TM, Roepstorff P, Mikkelsen JD (2000) Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from Aspergillus niger. Carbohydr Res 327:293–307CrossRefPubMedGoogle Scholar
  15. MacKinnon IM, Jardine WG, O’Kennedy N, Renard CM, Jarvis MC (2002) Pectic methyl and nonmethyl esters in potato cell walls. J Agric Food Chem 50:342–346CrossRefPubMedGoogle Scholar
  16. McCartney L, Ormerod AP, Gidley MJ, Knox JP (2000) Temporal and spatial regulation of pectic (1–>4)-beta-d-galactan in cell walls of developing pea cotyledons: implications for mechanical properties. Plant J 22:105–113CrossRefPubMedGoogle Scholar
  17. McCartney L, Steele-King CG, Jordan E, Knox JP (2003) Cell wall pectic (1–>4)-beta-d-galactan marks the acceleration of cell elongation in the Arabidopsis seedling root meristem. Plant J 33:447–454CrossRefPubMedGoogle Scholar
  18. McKie VA, Black GW, Millward-Sadler SJ, Hazlewood GP, Laurie JI, Gilbert HJ (1997) Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo-mode of action. Biochem J 323:547–555PubMedGoogle Scholar
  19. Obro J, Harholt J, Scheller HV, Orfila C (2004) Rhamnogalacturonan I in Solanum tuberosum tubers contains complex arabinogalactan structures. Phytochemistry 65:1429–1438CrossRefPubMedGoogle Scholar
  20. O’Shea MG, Samuel MS, Konik CM, Morell MK (1998) Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labeling and high-resolution separation. Carbohydr Res 307:1–12CrossRefGoogle Scholar
  21. Pauly M, Albersheim P, Darvill A, York WS (1999) Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants. Plant J 20:629–639CrossRefPubMedGoogle Scholar
  22. Pauly M, Eberhard S, Albersheim P, Darvill A, York WS (2001) Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells. Planta 214:67–74PubMedGoogle Scholar
  23. Peng L, Hocart CH, Redmond JW, Williamson RE (2000) Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production. Planta 211:406–414CrossRefPubMedGoogle Scholar
  24. Prime TA, Sherrier DJ, Mahon P, Packman LC, Dupree P (2000) A proteomic analysis of organelles from Arabidopsis thaliana. Electrophoresis 21:3488–3499CrossRefPubMedGoogle Scholar
  25. Proctor MR, Taylor EJ, Nurizzo D, Turkenburg JP, Lloyd RM, Vardakou M, Davies GJ, Gilbert HJ (2005) Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A. Proc Natl Acad Sci USA 102:2697–2702CrossRefPubMedGoogle Scholar
  26. Reiter WD, Chapple C, Somerville CR (1993) Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis. Science 261:1032–1035PubMedCrossRefGoogle Scholar
  27. Ridley BL, O’Neill MA, Mohnen D (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57:929–967CrossRefPubMedGoogle Scholar
  28. Wee EG, Sherrier DJ, Prime TA, Dupree P (1998) Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10:1759–1768CrossRefPubMedGoogle Scholar
  29. Willats WG, Steele-King CG, McCartney L, Orfila C, Marcus SE, Knox JP (2000) Making and using antibody probes to study plant cell walls. Plant Physiol Biochem 38:27–36CrossRefGoogle Scholar
  30. Willats WG, McCartney L, Knox JP (2001) In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana. Planta 213:37–44CrossRefPubMedGoogle Scholar
  31. Zablackis E, Huang J, Müller B, Darvill AG, Albersheim P (1995) Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves. Plant Physiol 107:1129–1138CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Christopher J. Barton
    • 1
  • Louise E. Tailford
    • 1
    • 3
  • Helen Welchman
    • 1
    • 4
  • Zhinong Zhang
    • 1
  • Harry J. Gilbert
    • 2
  • Paul Dupree
    • 1
  • Florence Goubet
    • 1
  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK
  2. 2.Institute for Cell and Molecular BiosciencesUniversity of Newcastle upon Tyne, The Medical SchoolNewcastle upon TyneUK
  3. 3.Institute for Cell and Molecular BiosciencesUniversity of Newcastle upon Tyne, The Medical SchoolNewcastle upon TyneUK
  4. 4.Drug Control Centre, King’s College LondonLondonUK

Personalised recommendations