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Control of xyloglucan endotransglucosylase activity by salts and anionic polymers

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Abstract

Crude extracts of cauliflower florets had high xyloglucan endotransglucosylase (XET) activity, but this was largely lost after partial purification and de-salting. Activity was restored (promoted up to 40-fold) by any of a wide variety of inorganic and organic salts. Optimum concentrations for Na+, K+ and NH4 + salts were typically ~300 mM. The chlorides of Ca2+, Mg2+, Al3+ and La3+ were optimally active at lower concentrations (e.g. 0.1 mM LaCl3), but became inhibitory at higher concentrations (e.g. 5 mM LaCl3). Some anionic polysaccharides at 0.04–0.2% w/v (e.g. gum arabic, pectin and hypochlorite-oxidised xyloglucan) promoted the XET activity of de-salted enzyme, especially if a sub-optimal concentration of NaCl was also present; others (e.g. homogalacturonan, 4-O-methyl-glucuronoxylan and alginate) were inhibitory. Similar ionic effects were noted on the XET activity of the Arabidopsis protein XTH24 (heterologously expressed by insect cells); in this case carboxymethylcellulose was also stimulatory. To look for endogenous modulators of XET activity, we prepared a cold-water extract of cauliflower florets; after boiling and centrifugation, the supernatant [boiled cauliflower preparation (BCP)] promoted the XET activity of de-salted cauliflower enzyme and of XTH24. About half the activator present in BCP was an ethanol-precipitable, anionic polymer of apparent Mr <5,000. After acid hydrolysis the polymer yielded much arabinose and galactose, and small amounts of galacturonic and glucuronic acids amino acids were also present. The polymer may thus contain arabinogalactan-proteins. We suggest that acidic polymers and/or other apoplastic ions are naturally occurring regulators of XET action in vivo, and may thus control cell wall assembly, loosening, and growth.

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Abbreviations

AGP :

Arabinogalactan-protein

BCP :

Boiled cauliflower preparation (cold-water-extract of cauliflower florets that was then boiled)

CMC :

Carboxymethylcellulose

DE :

Degree of esterification

GalA :

Galacturonic acid

GlcA :

Glucuronic acid

K av :

Elution volume relative to those of Blue Dextran (K av=0) and glucose (K av=1)

TFA :

Trifluoroacetic acid

V 0 :

Void volume (centre of elution peak of Blue Dextran)

V i :

Totally included volume (centre of elution peak of glucose)

XEH :

Xyloglucan endohydrolase (activity)

XET :

Xyloglucan endotransglucosylase (activity)

XLLGol :

A xyloglucan-derived oligosaccharide, xylose3·glucose3·galactose2·glucitol

XTH :

Xyloglucan endotransglucosylase/hydrolase (protein)

µ :

Ionic strength

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Acknowledgements

We are very grateful to Dr. Janet Braam (Rice University, Houston, Texas) for providing the XTH24 preparation. T.T. thanks the Japan Society for Promotion of Science (JSPS) for the award of a Fellowship. We thank the Biotechnology and Biological Sciences Research Council (UK) for financial support.

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  1. Takumi Takeda

    Present address: Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802, USA

Authors and Affiliations

  1. The Edinburgh Cell Wall Group, ICMB, The University of Edinburgh, Daniel Rutherford Building, The King’s Buildings, Edinburgh, EH9 3JH, UK

    Takumi Takeda & Stephen C. Fry

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  1. Takumi Takeda
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Correspondence to Stephen C. Fry.

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Takeda, T., Fry, S.C. Control of xyloglucan endotransglucosylase activity by salts and anionic polymers. Planta 219, 722–732 (2004). https://doi.org/10.1007/s00425-004-1267-9

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