Functional and chemical characterisation of XAF: a heat-stable plant polymer that activates xyloglucan endotransglucosylase/hydrolase (XTH)

Background and aims: Xyloglucan endotransglucosylase/hydrolase (XTH) proteins that possess xyloglucan endotransglucosylase (XET) activity contribute to cell-wall assembly and remodelling, orchestrating plant growth and development. Little is known about in-vivo XET regulation, other than at the XTH transcriptional level. Plants contain ‘cold-water-extractable, heat-stable polymers’ (CHPs) which are XTH-activating factors (XAFs) that desorb and thereby activate wall-bound XTHs. Since XAFs may control cell-wall modification in vivo, we have further explored their nature.
Methods: Material was cold-water-extracted from 25 plant species; proteins were precipitated by heat-denaturation, then CHP was ethanol-precipitated. For XAF assays, CHP (or sub-fractions thereof) was applied to washed Arabidopsis thaliana cell walls, and enzymes thus solubilised were assayed radiochemically for XET activity. In some experiments, the CHP was pre-treated with trifluoroacetic acid (TFA), alkali (NaOH) or glycanases.
Key results:
   · CHP specifically desorbed wall-bound XTHs, but not β-glucosidases, phosphatases or peroxidases.

·        · CHP preparations from 25 angiosperms all possessed XAF activity but had no consistent monosaccharide composition.

          · Of eleven individual plant polymers tested, only gum arabic and tamarind xyloglucan were XAF-active, albeit less so than CHP.

          · On gel-permeation chromatography, XAF-active cauliflower CHP eluted with molecular weight ~7,000–140,000, though no specific sugar residue(s) co-eluted exactly with XAF activity.

         · Cauliflower XAF activity survived cold alkali and warm dilute TFA (which break ester and glycofuranosyl linkages respectively), but was inactivated by hot 2M TFA (which breaks glycopyranosyl linkages).

        · Cauliflower XAF activity was remarkably stable to diverse glycanases and glycosidases.

Conclusions: XAFs are naturally occurring heat-stable polymers that specifically desorb (thereby activating) wall-bound XTHs. Their XAF activity considerably exceeds that of gum arabic and tamarind xyloglucan, and they were not identifiable as any major plant polysaccharide. We propose that XAF is a specific, minor, plant polymer that regulates xyloglucan transglycosylation in vivo, and thus wall assembly and restructuring