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Xyloglucan−pectin linkages are formed intra-protoplasmically, contribute to wall-assembly, and remain stable in the cell wall

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Abstract

We tested two hypotheses for the mechanism by which xyloglucan–pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using α-(1→5)-l-[3H]arabino- or β-(1→4)-d-[3H]galacto-oligosaccharides as model acceptor substrates. The 3H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [3H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [3H]Xyloglucan–pectin complexes were detectable <4 min after [3H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan–pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [3H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [3H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [3H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan–pectin bonds are stable and the pectic moiety aids wall-assembly.

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Abbreviations

Ara8-ol:

α-(1→5)-l-Arabino-octaitol

Gal8-ol:

β-(1→4)-d-Galacto-octaitol

EAW:

ethyl acetate/acetic acid/water (10:5:6 by vol.)

PCW:

primary cell wall

RG-I:

rhamnogalacturonan-I

TFA:

trifluoroacetic acid

XET:

xyloglucan endotransglucosylase [activity]

XTH:

xyloglucan endotransglucosylase/hydrolase [protein]

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Acknowledgements

We thank Dr Peter Dominy (University of Glasgow) for the kind provision of Arabidopsis cell-suspension cultures; Dr Takumi Takeda (Iwate Biotechnology Research Centre, Narita Kitakami Iwate, Japan) for the kind provision of pea xyloglucan; and Phillip Melling and Jack Cavers for preliminary work on the transglucosylation assays. We thank the BBSRC for funding this project.

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  1. Zoë A. Popper

    Present address: The Department of Botany, National University of Ireland, Galway, Ireland

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  1. The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King’s Buildings, Edinburgh, EH9 3JH, UK

    Zoë A. Popper & Stephen C. Fry

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  1. Zoë A. Popper
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Correspondence to Stephen C. Fry.

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Popper, Z.A., Fry, S.C. Xyloglucan−pectin linkages are formed intra-protoplasmically, contribute to wall-assembly, and remain stable in the cell wall. Planta 227, 781–794 (2008). https://doi.org/10.1007/s00425-007-0656-2

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