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Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

ABSTRACT: Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo.

SUBMITTER: Sharples SC

PROVIDER: S-EPMC5669584 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

Sharples Sandra C SC Nguyen-Phan Tu C TC Fry Stephen C SC

Journal of plant physiology 20170805

Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allos ...[more]

PMID: 28822306

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Project description:Background and aimsAlthough xyloglucans are ubiquitous in land plants, they are less abundant in Poales species than in eudicotyledons. Poales cell walls contain higher levels of ?-1,3/1,4 mixed-linked glucans and arabinoxylans than xyloglucans. Despite the relatively low level of xyloglucans in Poales, the xyloglucan endotransglucosylase/hydrolase (XTH) gene family in rice (Oryza sativa) is comparable in size to that of the eudicotyledon Arabidopsis thaliana. This raises the question of whether xyloglucan is a substrate for rice XTH gene products, whose enzyme activity remains largely uncharacterized.MethodsThis study focused on OsXTH19 (which belongs to Group IIIA of the XTH family and is specifically expressed in growing tissues of rice shoots), and two other XTHs, OsXTH11 (Group I/II) and OsXTH20 (Group IIIA), for reference, and measurements were made of the enzymatic activities of three recombinant rice XTHs, i.e. OsXTH11, OsXTH20 and OsXTH19.Key resultsAll three OsXTH gene products have xyloglucan endohydrolase (XEH, EC 3·2·1·151) activity, and OsXTH11 has both XEH and xyloglucan endotransglycosylase (XET, EC 2·4·1207) activities. However, these proteins had neither hydrolase nor transglucosylase activity when glucuronoarabinoxylan or mixed-linkage glucan was used as the substrate. These results are consistent with histological observations demonstrating that pOsXTH19::GUS is expressed specifically in the vicinity of tissues where xyloglucan immunoreactivity is present. Transgenic rice lines over-expressing OsXTH19 (harbouring a Cauliflower Mosaic Virus 35S promoter::OsXTH19 cDNA construct) or with suppressed OsXTH19 expression (harbouring a pOsXTH19 RNAi construct) did not show dramatic phenotypic changes, suggesting functional redundancy and collaboration among XTH family members, as was observed in A. thaliana.ConclusionsOsXTH20 and OsXTH19 act as hydrolases exclusively on xyloglucan, while OsXTH11 exhibits both hydrolase and XET activities exclusively on xyloglucans. Phenotypic analysis of transgenic lines with altered expression of OsXTH19 suggests that OsXTH19 and related XTH(s) play redundant roles in rice growth.

Project description: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. Because 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 the enzymes thus solubilized 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 ?-glucosidases, phosphatases or peroxidases. CHP preparations from 25 angiosperms all possessed XAF activity but had no consistent monosaccharide composition. Of 11 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 a molecular weight of ~7000-140 000, although 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 2 m 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.

Dataset Information

Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

Publications

Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

Similar Datasets

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