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Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

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Abstract

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (Kd) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

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Abbreviations

AL:

Micro-dissected aleurone layer

DiFA:

Diferulic acid

EM:

Electron microscopy

FA:

Ferulic acid

IB:

Inner bran

LM:

Light microscopy

OB:

Outer bran

UV:

Ultraviolet

WB:

Wheat bran

XYL11-WT:

(1→ 4)-β-endoxylanase (EC 3.2.1.8)

XYL11-INAC:

Inactive catalytic mutant of XYL11-WT

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Acknowledgements

We gratefully thank Dr. Fabienne Guillon (URPOI, INRA, Nantes) for the gift of anti (1→ 4)-β-unsubstituted xylan antiserum. This work was supported by a grant from the Champagne-Ardenne regional authorities. Electron microscopy observations were performed at the Centre de microscopie électronique à transmission conventionnelle of University of Paris VI, France.

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Authors and Affiliations

  1. Institut National de la Recherche Agronomique, UMR614 FARE, 2, esplanade Roland Garros, BP 224, 51686, Reims Cedex 02, France

    Johnny Beaugrand, Gabriel Paës, Philippe Debeire, Michael O’Donohue & Brigitte Chabbert

  2. Laboratoire de Pathologie Végetale, Institut National Agronomique-Paris-Grignon, 16, rue Claude Bernard, 75232, Paris Cedex 05, France

    Danièle Reis

  3. Centre National de la Recherche Scientifique, Université de Nantes, UMR6204, 2, rue de la Houssiniere, 44322, Nantes Cedex 03, France

    Masayuki Takahashi

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  1. Johnny Beaugrand
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  2. Gabriel Paës
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  4. Masayuki Takahashi
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  7. Brigitte Chabbert
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Correspondence to Brigitte Chabbert.

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Beaugrand, J., Paës, G., Reis, D. et al. Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase. Planta 222, 246–257 (2005). https://doi.org/10.1007/s00425-005-1538-0

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  • DOI: https://doi.org/10.1007/s00425-005-1538-0

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