Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule.

Xyloglucans are the major component of plant cell walls and bind tightly to the surface of individual cellulose microfibrils, thereby cross-linking them into a complex polysaccharide network of the cell wall. The cleavage and reconnection of xyloglucan cross-links are considered to play the leading role during chemical processes essential for wall expansion and, therefore, cell growth and differentiation. Although it is hypothesized that some transglycosylation is involved in these chemical processes, the enzyme responsible for the reaction was not identified. We have now purified a novel class of endo-type glycosyltransferase to apparent homogeneity from the extracellular space or the cell wall of the epicotyls of Vigna angularis, a bean plant. The enzyme is a glycoprotein with a molecular mass of about 33 kDa. The enzyme catalyzes both 1) endo-type splitting of a xyloglucan molecule and 2) linking of a newly generated reducing end of the xyloglucan to the nonreducing end of another xyloglucan molecule, thereby mediating the transfer of a large segment of the xyloglucan to another xyloglucan molecule. The transferase exhibits no glycosidase or glycanase activity. Substrate specificity of the enzyme was investigated using several polysaccharides with different glycosidic linkages as donor substrates and pyridylamino oligosaccharides as acceptor substrates, in which the reducing end of the carbohydrate was tagged with a fluorescent group. The enzyme required a basic xyloglucan structure, i.e. a beta-(1-->4)-glucosyl backbone with xylosyl side chains, for both acceptor and donor activity. Galactosyl or fucosyl side chains on the main chain were not required for the acceptor activity. The enzyme exhibited higher reaction rates when xyloglucans with higher M(r) were used as donor substrates. Xyloglucans smaller than 10 kDa were no longer the donor substrate. On the other hand, pyridylamino heptasaccharide acted as a good acceptor as did xyloglucan polymers. Based on these results we propose to designate this novel enzyme a xyloglucan: xyloglucano-transferase, to be abbreviated endo-xyloglucan transferase (EXT) or xyloglucan recombinase. This enzyme is the first enzyme identified that mediates the transfer of a high M(r) segment between polysaccharide molecules to generate chimeric polymers. We conclude that endo-xyloglucan transferase functions as a reconnecting enzyme for xyloglucans and is involved in the interweaving or reconstruction of cell wall matrix, which is responsible for chemical creepage that leads to morphological changes in the cell wall.     

A Barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3;1,4)-beta-D-glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.     

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls

A new type of xyloglucan-degrading enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The enzyme was purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The purified enzyme gave a single protein band of 33 kDa on SDS-PAGE. The enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the purified enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the purified enzyme. The purified enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.     

Probing expansin action using cellulose/hemicellulose composites

Cellulose-based composite materials containing xyloglucans or mannan-based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical alpha-expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose-only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose-only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1-4) beta-glucan to (1-4) beta-glucan hydrogen bonded contacts can also serve as substrates.     

The Effect of Xyloglucans on the Degradation of Cell-Wall-Embedded Cellulose by the Combined Action of Cellobiohydrolase and Endoglucanases from Trichoderma viride

Two endoglucanases of Trichoderma viride, endoI and endoIV, were assayed for their activity toward alkali-extracted apple xyloglucans. EndoIV was shown to have a 60-fold higher activity toward xyloglucan than endoI, whereas carboxymethyl cellulose and crystalline cellulose were better substrates for the latter. The enzymic degradation of cellulose embedded in the complex cell-wall matrix of apple fruit tissue has been studied using cellobiohydrolase (CBH) and these two different endoglucanases. A high-performance liquid chromatographic method (Aminex HPX-22H) was used to monitor the release of cellobiose and oligomeric xyloglucan fragments. Synergistic action between CBH and endoglucanases on cell-wall-embedded cellulose was, with respect to their optimal ratio, slightly different from that reported for crystalline cellulose. The combination of endoIV and CBH solubilized twice as much cellobiose compared to a combination of endoI and CBH. Apparently, the concomitant removal of the xyloglucan coating from cellulose microfibrils by endoIV is essential for an efficient degradation of cellulose in a complex matrix. Cellulose degradation slightly enhanced the solubilization of xyloglucans. These results indicate optimal degradation of cell-wall-embedded cellulose by a three-enzyme system consisting of an endoglucanase with high affinity toward cellulose (endoI), a xyloglucanase (endoIV), and CBH.     

In vivo colocalization of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of Arabidopsis roots

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO-SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO-SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide-SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO-SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.     

Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants

Xyloglucan is a well-characterized hemicellulosic polysaccharide that is present in the cell walls of all seed-bearing plants. The cell walls of avascular and seedless vascular plants are also believed to contain xyloglucan. However, these xyloglucans have not been structurally characterized. This lack of information is an impediment to understanding changes in xyloglucan structure that occurred during land plant evolution. In this study, xyloglucans were isolated from the walls of avascular (liverworts, mosses, and hornworts) and seedless vascular plants (club and spike mosses and ferns and fern allies). Each xyloglucan was fragmented with a xyloglucan-specific endo-glucanase and the resulting oligosaccharides then structurally characterized using NMR spectroscopy, MALDI-TOF and electrospray mass spectrometry, and glycosyl-linkage and glycosyl residue composition analyses. Our data show that xyloglucan is present in the cell walls of all major divisions of land plants and that these xyloglucans have several common structural motifs. However, these polysaccharides are not identical because specific plant groups synthesize xyloglucans with unique structural motifs. For example, the moss Physcomitrella patens and the liverwort Marchantia polymorpha synthesize XXGGG- and XXGG-type xyloglucans, respectively, with sidechains that contain a beta-D-galactosyluronic acid and a branched xylosyl residue. By contrast, hornworts synthesize XXXG-type xyloglucans that are structurally homologous to the xyloglucans synthesized by many seed-bearing and seedless vascular plants. Our results increase our understanding of the evolution, diversity, and function of structural motifs in land-plant xyloglucans and provide support to the proposal that hornworts are sisters to the vascular plants.     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.      

Structural analysis of xyloglucans in the primary cell walls of plants in the subclass Asteridae

The structures of xyloglucans from several plants in the subclass Asteridae were examined to determine how their structures vary in different taxonomic orders. Xyloglucans, solubilized from plant cell walls by a sequential (enzymatic and chemical) extraction procedure, were isolated, and their structures were characterized by NMR spectroscopy and mass spectrometry. All campanulids examined, including Lactuca sativa (lettuce, order Asterales), Tenacetum ptarmiciflorum (dusty miller, order Asterales), and Daucus carota (carrot, order Apiales), produce typical xyloglucans that have an XXXG-type branching pattern and contain alpha-d-Xylp-, beta-D-Galp-(1-->2)-alpha-D-Xylp-, and alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xylp- side chains. However, the lamiids produce atypical xyloglucans. For example, previous analyses showed that Capsicum annum (pepper) and Lycopersicon esculentum (tomato), two species in the order Solanales, and Olea europaea (olive, order Lamiales) produce xyloglucans that contain arabinosyl and galactosyl residues, but lack fucosyl residues. The XXGG-type xyloglucans produced by Solanaceous species are less branched than the XXXG-type xyloglucan produced by Olea europaea. This study shows that Ipomoea pupurea (morning glory, order Solanales), Ocimum basilicum (basil, order Lamiales), and Plantago major (plantain, order Lamiales) all produce xyloglucans that lack fucosyl residues and have an unusual XXGGG-type branching pattern in which the basic repeating core contains five glucose subunits in the backbone. Furthermore, Neruim oleander (order Gentianales) produces an XXXG-type xyloglucan that contains arabinosyl, galactosyl, and fucosyl residues. The appearance of this intermediate xyloglucan structure in oleander has implications regarding the evolutionary development of xyloglucan structure and its role in primary plant cell walls.     

Purification of Xyloglucan Hydrolase/Endotransferase from Cell Walls of Azuki Bean Epicotyls

An enzyme involved in the breakdown of xyloglucans was purifiedfrom an extract of cell walls of azuki bean epicotyls obtainedwith 1 M NaCl and purified by column chromatography on severaldifferent resins. The purified enzyme gave a single band ofa protein with a molecular mass of about 32 kDa after SDS-PAGE.The enzyme hydrolyzed the xyloglucans of high molecular massfrom azuki cell walls to yield fragments of about 50 kDa withoutproduction of any oligo- or monosaccharides. Moreover, the enzymehad hardly any effect on xyloglucans of less than 60 kDa. Theenzyme also hydrolyzed xyloglucans from tamarind, but it didnot react with cellulose derivatives. In the presence of pyridylamino-labeledxyloglucan oligosac-charides as acceptor substrates, the enzymecatalyzed the transfer of 50-kDa products to the oligosaccharides.The K_m value of the enzyme for xyloglucans of 540 kDa was similarin the presence and in the absence of xyloglucan oligosaccharidesas acceptors: 1.0 mg ml^–1. These results suggest thatthe enzyme was an endotransferase but had unusual acceptor specificity,preferring smaller acceptors such as water. (Received September 9, 1996; Accepted March 16, 1997)     

In vitro molecular weight increase in xyloglucans by an apoplastic enzyme preparation from epicotyls of Vigna angularis

In order to gain insight into the mechanism of cell extension growth, enzymic processes involved in structural modification of cell wall xyloglucans were investigated, using an apoplastic enzyme preparation from epicotyls of dark grown Vigna angularis Ohwi et Ohashi cv. Takara and purified xyloglucans derived from cell walls of Vigna. The reaction of Vigna xyloglucan (mass average molecular weight=420 kDa) with the apoplastic enzyme preparation gave three fractions: (1) a waterinsoluble high molecular weight (820 kDa) xyloglucan fraction (WI), (2) a watersoluble low molecular weight (149 kDa) xyloglucan fraction (WS), and (3) an 80% ethanol-soluble monosaccharide fraction (ES). WI and WS were chiefly composed of t -galactosyl-, t -xylosyl-, 2-xylosyl-, 4-glucosyl- and 4,6-glucosyl residues, whereas ES was composed of fucose, galactose, glucose and xylose monomers. The data indicate that WI is generated by the linking of xyloglucan molecules by some alkali stable linkages, probably of glycosidic nature. The optimal pH for the WI-producing activity of the apoplastic enzyme preparation was 5.4. Higher WI-producing activity was detected in the upper juvenile than in the lower non-elongating regions of the epicotyl. Our data suggest the possible involvement of a transglycosylation reaction in the structural changes of the xyloglucans that are responsible for cell extension growth of the Vigna angularis epicotyl. The data are also consistent with the idea that the enzymic processes are regulated by hydrogen ions in the apoplastic space.     

Xyloglucan in the primary cell wall: assessment by FESEM,selective enzyme digestions and nanogold affinity tags

Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load‐bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high‐resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase‐digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan‐specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold‐conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan‐depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.     

Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.     

Bilberry xyloglucan--novel building blocks containing beta-xylose within a complex structure

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.     

Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites

Background

Xyloglucan xyloglucosyl transferases (EC 2.4.1.207), known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates.

Methods

cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.

Results

Significant differences in catalytic efficiencies (k_cat·K_m⁻¹) were observed and these values were 0.01, 0.02 and 0.2 s⁻¹·mg⁻¹·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations.

Conclusions

The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.

General significance

The results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents.     

Structural analyses of two arabinose containing oligosaccharides derived from olive fruit xyloglucan: XXSG and XLSG

Xyloglucan oligosaccharides were prepared by endo-(1-->4)-beta-D-glucanase digestion of alkali-extractable xyloglucan from olive fruit and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography and high-performance anion-exchange chromatography. The two most abundant oligosaccharides were converted to the corresponding oligoglycosyl alditols by borohydride reduction and structurally characterised by NMR spectroscopy and post-source decay (PSD) fragment analysis of matrix-assisted laserinduced desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. The results revealed that olive fruit xyloglucan is mainly built from two novel oligosaccharides: XXSG and XLSG. The structure of the oligosaccharides confirmed the presence of a specific xyloglucan in olive fruit with alpha-L-Araf-(1-->2)-alpha-D-Xylp sidechains as was suggested previously. The presence of such sidechains is a common feature of xyloglucans with an XXGG core produced by solanaceous plants but has not been demonstrated for other dicotyledonous plants, which have in general an XXXG core. Direct treatment of cell wall material from olive fruit with pectin degrading enzymes in combination with endo-(1-->4)-beta-D-glucanase revealed that some of the arabinose residues of the oligosaccharides XXSG and XLSG are substituted with either 1 or 2 O-acetyl groups.     

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50mm NaOH at 1°, m KOH at 1° and 20°, and 4m KOH at 20°, to leave a residue of α-cellulose. From the 4m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.     

Xyloglucan structure and post-germinative metabolism in seeds of Copaifera langsdorfii from savanna and forest populations

The cotyledons of Copaifera langsdorfii Desf, have been shown to contain a water-soluble xyloglucan (amyloid), which represents about 40% of the seed's dry weight. On acid hydrolysis its composition (Glc:Xyl:Gal = 4.0:2.8–2.9:1.5–1.7) was similar to that of the well-characterized xyloglucan of Tamarindus indica L. (Glc:Xyl:Gal = 4.0:3.0–3.1:1.4). On hydrolysis with pure Trichoderma viride cellulase, both C. langsdorfii and T. indica xyloglucan gave the same xyloglucan oligosaccharides: but in significantly different proportions A:B₁:B₂:C = 1:0.4–0.5:2.1–2.2:3.1–3.4 in T. indica , and 1:1.1:1.8:7.4 and 1:1.3:2.6:12 for C. langsdorfii , savanna and forest populations respectively. This demonstrated a difference in fine molecular structure, notably in the distribution of the terminal non-reducing galactose substituents, between the xyloglucans of the two species and indicated differences in the specificities of their biosynthetic mechanisms. The xyloglucans obtained from C. langsdorfii seeds harvested from savanna and forest environments were slightly different, one from the other, in their sugar-residue composition (Glc:Xyl:Gal = 4.0:2.9:1.5 and 4.0:2.8:1.7, respectively), and were significantly different in the relative proportions of the xyloglucan oligosaccharides released on cellulase hydrolysis (above). Using light microscopy and biochemical methods, no difference in the pattern or rate of postgerminative xyloglucan metabolism was detected in seeds of savanna and forest origin. This is the first clear experimental evidence for differences in a storage xyloglucan structure between populations of the same species. It may indicate environmental influences on xyloglucan biosynthesis.