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Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

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Abstract

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.

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Abbreviations

Api:

d-Apiose

AceA:

l-Aceric acid

Dha:

2-Keto-3-deoxy-d-lyxo-heptulosaric acid

DMB:

1,2 Diamino-4,5-methylene dioxybenzene

Kdo:

2-Keto-3-deoxy-d-manno-octulosonic acid

ESI-MS:

Electrospray ionisation mass spectrometry

EPG:

Endo-α(1,4)-polygalacturonase

MALDI-TOF:

Matrix-assisted laser desorption ionisation-time of flight

OLIMP:

Oligosaccharide mass profiling method

2-OMeFuc:

2-O-Methyl l-fucose

2-OMeXyl:

2-O-Methyl d-xylose

RG-II:

Rhamnogalacturonan II

TFA:

Trifluoroacetic acid

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Acknowledgments

The Nottingham Arabidopsis Stock Center and Gregory Mouille, INRA Versailles, France are acknowledged for providing seeds for the Arabidopsis insertion lines and mutants. The authors thank Muriel Bardor, EA4358 University of Rouen, France for MALDI TOF analyses. This research was supported in France by the CNRS, the Région Haute-Normandie and the University of Rouen. Research at UPSC Sweden (A.M., A.-M.W.) was supported by the Swedish Foundation for Scientific Research (SSF) and Formas.

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Author notes

  1. Martial Séveno, Ai-Min Wu & Alan Marchant

    Present address: CNRS UMR 5203, Plate-forme de Protéomique Fonctionnelle, Institut de Génomique Fonctionnelle, IFR3, 141 Rue de la Cardonille, 34094, Montpellier Cedex 05, France

Authors and Affiliations

  1. EA 4358, IFRMP 23, University of Rouen, 76821, Mont Saint Aignan, France

    Martial Séveno, Aline Voxeur, Christophe Rihouey, Azeddine Driouich & Patrice Lerouge

  2. Department of Forest Genetics and Plant Physiology, 901-83, Umeå, Sweden

    Ai-Min Wu & Alan Marchant

  3. Forestry and Forest Products Research Institute, P. O. Box 16, Tsukuba Norin Kenkyu Danchi-nai, Ibaraki, 305-8687, Japan

    Tadashi Ishii

  4. INRA, Unité Mixte de Recherche 619 Biologie du Fruit, 33883, Villenave d’Ornon, France

    Christian Chevalier

  5. UR1268 Biopolymères Interactions Assemblages, 44300, Nantes, France

    Marie Christine Ralet

  6. School of Biological Sciences, University of Southampton, Southampton, UK

    Ai-Min Wu & Alan Marchant

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  1. Martial Séveno
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Correspondence to Patrice Lerouge.

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Séveno, M., Voxeur, A., Rihouey, C. et al. Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology. Planta 230, 947–957 (2009). https://doi.org/10.1007/s00425-009-0996-1

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