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An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls

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Abstract

Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.

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Abbreviations

CTAB:

Cetyltrimethylammonium bromide

WAX:

Wheat arabinoxylan

PAHBAH:

p-Hydroxybenzoic acid hydrazide

XET:

Xyloglucan endotransglycosylase

XTH:

Xyloglucan endotransglycosylase/hydrolase

Xyl2-ol:

Xylobiitol

Xyl3-ol:

Xylotriitol

Xyl4-ol:

Xylotetraitol

Xyl5-ol:

Xylopentaitol

Xyl6-ol:

Xylohexaitol

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Acknowledgments

We thank Erin O’Donoghue, William Laing and Robert Schaffer for comments on the manuscript. We are indebted to Lionel M. Hill (John Innes Centre, Norwich, England) for his advice with the enzyme kinetics. We also thank Cherie Walsh, ARC Centre of Excellence in Plant Cell Walls, The University of Melbourne, for technical assistance with cell wall methylation analyses. Antony Bacic acknowledges the support of the ARC Centre of Excellence in Plant Cell Walls. This project was funded by the Ministry of Business Innovation and Employment, New Zealand (contract C11X1007).

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Author notes

  1. Monto H. Kumagai

    Present address: XtremeSignPost Inc., Leucadia, CA, 92024, USA

Authors and Affiliations

  1. The New Zealand Institute for Plant and Food Research Limited, Mount Albert Research Centre, Private Bag 92169, Auckland, 1142, New Zealand

    Sarah L. Johnston, Roneel Prakash, Ross G. Atkinson & Roswitha Schröder

  2. Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI, 96822-2279, USA

    Nancy J. Chen, Monto H. Kumagai, Helen M. Turano & Robert E. Paull

  3. The New Zealand Institute for Plant and Food Research Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, 3240, New Zealand

    Janine M. Cooney

  4. ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Melbourne, VIC, 3010, Australia

    Roshan Cheetamun & Antony Bacic

  5. The New Zealand Institute for Plant and Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, 4442, New Zealand

    David A. Brummell

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  1. Sarah L. Johnston
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Correspondence to Roswitha Schröder.

Electronic supplementary material

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Table S1 Peptide fragments in fraction 16 eluted from the MonoS column (PDF 94.1 kb)

Table S2 Predicted xylanase proteins and open reading frames in the papaya genome (PDF 263 kb)

425_2012_1766_MOESM3_ESM.pdf

Fig. S1 Analysis of the recombinant protein products produced in Nicotiana benthamiana leaves using viral vector TTOSA1-XYL (PDF 172 kb)

Fig. S2 2D-gel electrophoresis and gel blot of papaya fruit proteins (PDF 256 kb)

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Johnston, S.L., Prakash, R., Chen, N.J. et al. An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls. Planta 237, 173–187 (2013). https://doi.org/10.1007/s00425-012-1766-z

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