Factors that affect the extraction of xyloglucan from the primary cell walls of suspension-cultured rose cells

Edelmann, Hans G.; Fry, Stephen C.

doi:10.1016/0008-6215(92)84135-f

Cited by 43 publications

(30 citation statements)

References 31 publications

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“…Radiolabelled rose cells (7 g fresh weight) were suspended (20 mg fresh weight/ml) in 6 M NaOH containing 1% (w/v) NaBH 4 and left shaking at 37°C for 24 h. This treatment has been shown to extract essentially all the xyloglucan and 3 H from the walls of cell-suspension cultures (Edelmann and Fry 1992). The alkali extract was ®ltered through a polypropylene frit (in an empty`PolyPrep' column; BioRad), acidi®ed by addition of glacial acetic acid (0.5 vols., added slowly with stirring on ice), dialysed, freeze-dried, and re-dissolved in 60 ml of 11 mM acetate buer, pH 4.7.…”

Section: Methodsmentioning

confidence: 99%

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

Thompson

Fry

2000

Planta

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169

102

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Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. 'Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on 'Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeatunits of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins.

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Section: Methodsmentioning

confidence: 99%

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

Thompson

Fry

2000

Planta

Self Cite

169

102

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“…The homogenate was immediately filtered on a Polyprep column and washed with 6i3 ml of sonication buffer ; the filtrate (' protoplast contents ') was stored frozen. The wall-rich residue was resuspended in 5 ml of 6 M NaOH containing 1 % w\v NaBH % [39] and shaken gently at 37 mC for 24 h. The suspension was then filtered on a Polyprep column and washed with 3i3 ml of NaOH\NaBH % solution ; the filtrate was acidified by slow addition of acetic acid, dialysed against water at 4 mC and stored at k20 mC (' hemicellulose extract ').…”

Section: Fractionation Of Cell Polysaccharidesmentioning

confidence: 99%

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate

Thompson

Smith

Fry

1997

Biochemical Journal

105

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Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a ≈ 2 h pulse of L-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C,3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a ≈ 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of ~ 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C,3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.

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“…Concentrated alkali, which causes microfibril swelling, is required to extract xyloglucan from the wall (Chambat eta/., 1984;Edelmann and Fry, 1992;Hayashi and Maclachlan, 1984). In muro, xyloglucan is proposed to form a monolayer coating the surface of microfibriis and also to penetrate the microfibrils in areas of high amorphous content (Hayashi et al, 1987;Valent and Albersheim, 1974).…”

Section: Introductionmentioning

confidence: 99%

In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects

et al. 1995

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SummaryFeatures of the interaction between cellulose and xyloglucen have been studied using the cellulose-producing bacterium Acetobacter aceti ssp. xylinum (ATCC 53524) and tamarind seed xyloglucen. Direct microscopic evidence is provided for the generation of cross-bridges between cellulose ribbons produced in the presence of xyloglucan but not cerboxymathyiceltulose. Cross-bridge lengths are very similar to those observed for de-pectinated onion cell walls. Similar cross-bridge lengths are observed following mixing of isolated A. xylinum cellulose and xyloglucan, showing that network formation can be an abiotic process. The level of incorporation of xyloglucan in an actively growing system (ca. 38% of cellulose) is an order of magnitude higher than that observed in mixtures of isolated polymers and is comparable with cell wall levels.NMR spectroscopy suggests that 80-85% of incorporated xyloglucen is segmentally rigid with the backbone adopting an extended 'cellulosic' conformation and probably aligned with cellulose chains. The remaining xyioglucan is more mobile and is assigned to cross-bridges with, on average, a twisted backbone conformation. No evidence for specific involvement of side-chain residues in binding is found, and the observation of cross-bridges with a non-fucosylated xyloglucan shows that fucose residues are not essential for network formation. Xyloglucen causes cellulose ribbons to become more amorphous end to have a decreased IoJl[~ crystallite ratio without any significant alteration in ribbon diameter.Based on the findings that levels of xyloglucan incorporation, the presence and lengths of cross-bridges, and the modification of cellulosic molecular organization are all similar to those found in plant cell walls, we suggest that

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Factors that affect the extraction of xyloglucan from the primary cell walls of suspension-cultured rose cells

Cited by 43 publications

References 31 publications

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate

In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects

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