Search

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207 [EC] ) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151 [EC] ) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage β-glucan. Isoforms differed in optimum pH (5.07.5), in temperature dependence and in acceptor substrate preferences.

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Dehydroascorbate and traces of ascorbate were present apoplastically in living spruce (Picea abies) twigs. Since the proposed apoplastic ascorbate degradation pathway contains several steps that possibly generate H(2)O(2), the effects of ascorbate and some of its degradation products were tested on apoplastic H(2)O(2) concentrations in a cell culture of P. abies as a model and on non-enzymic H(2)O(2) production in vitro. Ascorbate scavenged H(2)O(2) in the culture medium of lignin-producing Picea cells and in spent and boiled spent medium; in the presence of Cu(2+) or fresh medium, ascorbate led to the non-enzymic generation of H(2)O(2). Preparations of dehydroascorbate (the initial oxidation-product of ascorbate), and diketogulonate (the hydrolysis-product of dehydroascorbate) induced H(2)O(2) accumulation both non-enzymically and enzymically in Picea cell-suspensions. Paper electrophoresis showed that the dehydroascorbate and diketogulonate preparations contained several degradation products; some of these probably contributed to H(2)O(2) production and/or scavenging in these experiments, and would also do so in vivo. These results indicate a complex ability of apoplastic ascorbate, dehydroascorbate, diketogulonate, and further products to modulate H(2)O(2) concentrations, with potential consequences for the control of growth, development and lignification.

In this work the effect of injection of xyloglucan-derived oligosaccharides (XGOs) into whole persimmon (Diospyros kaki L.) fruits on ethylene biosynthesis was investigated. Fruits collected during different ripening stages produced low levels of ethylene without a climacteric-like peak. Pretreatment of these fruits with 10 cm 3 C 2 H4 m -3 for 8 h stimulated little or no endogenous ethylene production. However, when persimmon fruits were injected with a mixture of XGOs a burst in ethylene production was observed compared with water-injected control fruits or fruits injected with different monosaccharide solutions. In order to study the influence of oligosaccharide structure and fruit ripening stage on the ability of XGOs to induce ethylene synthesis, fucosylated and non-fucosylated XGOs were injected into persimmon fruits harvested at two different ripening stages. Both oligosaccharide structures were able to induce ethylene production. Induction of ethylene by XGOs was much more evident in fruits harvested later in time, indicating that the process is developmentally regulated. The levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in injected persimmon fruits were also examined. This study showed that the increase in the rate of ethylene biosynthesis induced by XGOs was accompanied by the accumulation of its metabolic precursor ACC.

The plant cell-wall matrix is equipped with more than 20 glycosylhydrolase activities, including both glycosidases and glycanases (exo- and endo-hydrolases, respectively), which between them are in principle capable of hydrolysing most of the major glycosidic bonds in wall polysaccharides. Some of these enzymes also participate in the 'cutting and pasting' (transglycosylation) of sugar residues-enzyme activities known as transglycosidases and transglycanases. Their action and biological functions differ from those of the UDP-dependent glycosyltransferases (polysaccharide synthases) that catalyse irreversible glycosyl transfer. Based on the nature of the substrates, two types of reaction can be distinguished: homo-transglycosylation (occurring between chemically similar polymers) and hetero-transglycosylation (between chemically different polymers). This review focuses on plant cell-wall-localized glycosylhydrolases and the transglycosylase activities exhibited by some of these enzymes and considers the physiological need for wall polysaccharide modification in vivo. It describes the mechanism of transglycosylase action and the classification and phylogenetic variation of the enzymes. It discusses the modulation of their expression in plants at the transcriptional and translational levels, and methods for their detection. It also critically evaluates the evidence that the enzyme proteins under consideration exhibit their predicted activity in vitro and their predicted action in vivo. Finally, this review suggests that wall-localized glycosylhydrolases with transglycosidase and transglycanase abilities are widespread in plants and play important roles in the mechanism and control of plant cell expansion, differentiation, maturation, and wall repair.

To study the metabolism of oligosaccharins, we monitored the fate of (6- 14 C)-labelled oligo-α-(1→4)-D-galacturonides of degree of polymerization 8 and 9 (GalA 8 and GalA 9 ) when administered at ∼1 μM to cell suspension cultures of 'Paul's Scarlet' rose (Rosa sp.). Neither GalA 8 nor GalA 9 was appreciably taken up by or bound to the cells. Both GalA 8 and GalA 9 were rapidly hydrolysed by the culture (half-life 2-4 h). The products were smaller oligogalacturonides and galacturonic acid, indicating the action of exo-polygalacturonase. It is suggested that exo-polygalacturonase plays an important role in controlling the concentrations of biologically-active oligogalacturonides in uninfected plant tissues.

The promotion of stem elongation in dwarf pea plants by GA3 has been correlated with an increase in extractable xyloglucan endotransglycosylase (XET) activity (Potter and Fry, 1993). Doses of auxin that caused either elongation or lateral swelling in pea stems have not been found to increase XET activity (Fry et al., 1992). We therefore explored the generality of the association between GA3 action and XET activity by testing three other bioassay systems. A similar correlation was found in the hypocotyls of intact lettuce seedlings, where GA3 strongly promoted elongation and doubled the extractable XET activity per unit fresh weight. In the hypocotyls of intact cucumber seedlings, GA3 evoked a prolonged promotion of elongation for at least 96 h; this was correlated with only a small increase in XET activity per unit fresh weight of tissue. IAA promoted elongation for only 48 h, but this effect was correlated with a larger increase in XET activity per unit fresh weight than that due to GA3. In cell suspension cultures of spinach, much of the XET activity was present in solution in the culture medium. GA3 had little effect on this fraction for the first 9 d, but thereafter the hormone suppressed a sudden burst in soluble extracellular XET activity that occurred in the untreated controls. A further proportion of the XET activity was ionically wall-bound; this fraction was enhanced by GA3 in the early phase of the growth cycle of the culture and inhibited in later phases. Pre-adaptation of the culture by growth for 6 years in the presence of 10−7 M GA3 intensified the response of the cells to re-addition of GA3. We conclude that there is no simple or unique relationship between total extractable XET activity and GA3 action. Nevertheless, we have extended the evidence that XET is often subject to the effects of GA3 in systems where this hormone influences growth.

Rhamnogalacturonan-II inhibited the uptake of [ 14 C]leucine and, consequently, the incorporation of [ 14 C]leucine into acid-precipitable proteins by suspension-cultured tomato cells. Fractionation of rhamnogalacturonan-II showed that the lower molecular components were the most effective. KDO and apiose, both constituents of rhamnogalacturonan-II, also inhibited [ 14 C]leucine incorporation weakly, suggesting that these sugar residues may be an integral requirement for the biological activity of rhamnogalacturonan-II

A series of physical and chemical analyses were made on the expanding zone of maize seedling roots grown in hydroponics. Comparison of longitudinal profiles of local relative elemental growth rate and turgor pressure indicated that cell walls become looser in the apical 5 mm and then tighten 5-10 mm from the root tip. Immersion of roots in 200 mol m-3 mannitol (an osmotic stress of 0-48 MPa) rapidly and evenly reduced turgor pressure along the whole growing region. Growth was reduced to a greater extent in the region 5-10 mm from the root tip than in the apical region. This indicated rapid wall-loosening in the root tip, but not in the more basal regions. Following 24 h immersion in 400 mol m-3 mannitol (an osmotic stress of 0-96 MPa) turgor had recovered to pre-stressed values. Under this stress treatment, growth was reduced in the region 4-10 mm from the root tip, despite the recovery of turgor, indicating a tightening of the wall. In the root apex, local relative elemental growth rate was unchanged in comparison to control tissue, showing that wall properties here were similar to the control values. Cellulose microfibrils on the inner face of cortical cell walls became increasingly more parallel to the root axis along the growth profile of both unstressed and stressed roots. Orientation did not correlate with the wall loosening in the apical region of unstressed roots, or with the tightening in the region 5-10 mm from the root tip following 24 h of osmotic stress. Longitudinal profiles of the possible wall-loosening enzyme xyloglucan endotransglycosylase (XET) had good correspondence with an increase in wall loosening during development. In the zone of wall tightening following osmotic stress, XET activity was decreased per unit dry weight (compared with the unstressed control), but not per unit fresh weight.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207 [EC] ) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151 [EC] ) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage â-glucan. Differences were found in pH- and temperature-dependence as well as in acceptorsubstrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.

15 pages, 9 figures.-- PMID: 18316315[PubMed].-- Supporting information available at: http://jxb.oxfordjournals.org/content/59/4/875/suppl/DC1, Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition., This work was funded by the Spanish ‘Ministerio de Ciencia y Tecnología’ (BIO2001-1140). VG was financed by a pre-doctoral grant from the ‘Generalitat de Catalunya’ (2003-FI00090). In addition, VG was funded by two grants from the Generalitat de Catalunya (2005-BE00104 and 2006-BE00668) for her work performed at Professor Fry's and Dr Ruel's laboratories. SF was financed by a post-doctoral grant from the ‘Generalitat de Catalunya’ (2003PIV-A-00033) and by an I3P contract from the ‘Consejo Superior de Investigaciones Científicas’. DC-R was financed by the Spanish ‘Ministerio de Educacion y Ciencia’ (‘Ramon y Cajal’ Program). This work was carried out within the framework of the ‘Xarxa de Referència en Biotecnologia’ from the ‘Generalitat de Catalunya’. SCF was funded by the UK Biotechnology and Biological Sciences Research Council. We are indebted to Dr Castresana (IBMB-CSIC) for his advice on the phylogenetic analyses, Dr Capellades for her technical support and the sequencing, and the greenhouse teams of IBMB-CSIC.

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.

The quantitatively major nonasaccharide (XG9) derived from xyloglucan by digestion with cellulase exhibits anti-auxin activity in the pea stem segment straight-growth bioassay; the most effective concentration of XG9 is c. 10~9 M. Previous work had shown that XG9 owes its biological activity to the presence of a terminal a-L-fucopyranose residue. In order to investigate to what extent the remainder of the XG9 molecule is essential for activity, several fucose-containing compounds were tested for their ability to mimic the anti-auxin effect of XG9. A fucose-containing pentasaccharide of xyloglucan (XG5; probable structure Fuc -> Gal -> Xyl ->■ Glc -> Glc) was, at 10~8 M, about as effective an anti-auxin as 10~9 M XG9; unlike XG9, XG5 did not diminish in effectiveness at 10~7 M. The human milk trisaccharide, 2'-fucosyl-lactose [i.-fucopyranosyl-a-(l ^2)-D-galactopyranosyl-/i-(l-> 4)-D-glucose], whose Fuc -> Gal unit is identical with that of XG9, inhibited auxin-induced elongation over a wide range of concentrations centred on about 10~8 M. 2'-Fucosyl-lactose at 10~8 M was about as effective an anti-auxin as 10~9 M XG9. Free L-fucose and methyl-a-L-fucopyranoside were unable to inhibit auxin-induced growth at any concentration tested (10~10 M to 10-6 M) and neither compound interfered with the inhibition caused by 10~9 M XG9 when co-incubated at concentrations up to 10~4 M. The results confirm the essential role of an «-linked terminal fucose residue in the anti-auxin activity of XG9 and show that the sub-terminal galactose residue may also be required. Possible reasons why high concentrations of XG9 fail to antagonize auxin-induced growth while high concentrations of XG5 and 2'-fucosyl-lactose continue to do so are discussed.

Xyloglucan is a polysaccharide found in the primary cell walls of all higher plants examined. Its cellulose-like backbone, which is about 015 to 1-5 ?un long, consists of 300 to 3000 /3-(l ^4)-linked D-glucopyranose residues. About 60-75% (or, in grasses, about 30-40%) of the glucose residues have side-chains attached to position 6. The major side-chains are: D-xylopyranosyl-a-1 ->, D-galactopyranosyl-^-(l ^2)-D-xylopyranosyl-a-l L-arabinofuranosyl-(l 2)-D-xylopyranosyl-a-1 and (except in grasses) L-fucopyranosyl-a-(l-»2)-D-galactopyranosyl-j3-(l-^-2)-D-xylopyranosyl-a-lThere is some regularity in the distribution of these side-chains along the backbone. Xyloglucan plays two very different roles in the control of cell growth: (a) as a major building material of the wall [concentration of xyloglucan in the wall in vivo ~ 10% (w/v)] it probably directly dictates wall extensibility and, therefore, the rate of cell expansion and (b) it can be broken down to a fucose-containing oligosaccharide which [at a concentration of ~ 0 0000001% (w/v)] exerts a hormone-like anti-auxin effect on growth. In addition, xyloglucan lacking fucose is used by certain dicotyledonous seeds as a food reserve which is mobilized after germination. Xyloglucan is, therefore, the subject of considerable current interest in several apparently disparate areas of botany.