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Xyloglucan endotransglucosylase activity loosens a plant cell wall.

ABSTRACT: BACKGROUND AND AIMS: Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in the wall so that the microfibrils can move relative to each other. Here the effect was studied of a purified recombinant xyloglucan endotransglucosylase/hydrolase (XTH) on the extension of isolated cell walls. METHODS: The epidermis of growing onion (Allium cepa) bulb scales is a one-cell-thick model tissue that is structurally and mechanically highly anisotropic. In constant load experiments, the effect of purified recombinant XTH proteins of Selaginella kraussiana on the extension of isolated onion epidermis was recorded. KEY RESULTS: Fluorescent xyloglucan endotransglucosylase (XET) assays demonstrate that exogeneous XTH can act on isolated onion epidermis cell walls. Furthermore, cell wall extension was significantly increased upon addition of XTH to the isolated epidermis, but only transverse to the net orientation of cellulose microfibrils. CONCLUSIONS: The results provide evidence that XTHs can act as cell wall-loosening enzymes.

SUBMITTER: Van Sandt VS

PROVIDER: S-EPMC2759230 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Xyloglucan endotransglucosylase activity loosens a plant cell wall.

Van Sandt Vicky S T VS Suslov Dmitry D Verbelen Jean-Pierre JP Vissenberg Kris K

Annals of botany 20071004 7

<h4>Background and aims</h4>Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in th ...[more]

PMID: 17916584

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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.

Project description:1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that it thus causes the wall-loosening required for plant cell expansion.

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Xyloglucan endotransglucosylase activity loosens a plant cell wall.

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Xyloglucan endotransglucosylase activity loosens a plant cell wall.

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