The cell wall-modifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction of tomato against the plant parasite Cuscuta reflexa.

A suppressive subtractive hybridization technique was used to identify genes, which were induced during the early phases of the interaction between dodder (Cuscuta reflexa), a phanerogamic parasite, and its incompatible host plant tomato. One of the identified genes encodes a tomato xyloglucan endotransglycosylase/hydrolase (XTH)--an enzyme involved in cell wall elongation and restructuring. The corresponding LeXTH1 mRNA accumulated 6 h after attachment of the parasite. In contrast, wounding did not influence the expression level. Subsequent to LeXTH1 mRNA accumulation, an increase in XTH activity at the infection sites as well as in adjacent tissues was observed. The effect of IAA on LeXTH1 expression was analyzed because the concentration of this phytohormone is known to increase in the tomato tissue during the interaction with the parasite. LeXTH1 mRNA accumulation was in fact induced by external application of auxin. However, in the auxin-insensitive tomato mutant diageotropica, Cuscuta induced LeXTH1-mRNA accumulated with a time course similar to wild type tomato. Thus, auxin appears not to be an essential signal for infection-induced LeXTH1 activation. Our data suggest a role for xyloglucan transglycosylation in defence reactions associated with the incompatible tomato- Cuscuta interaction.

Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays.

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.

Root hair initiation is coupled to a highly localized increase of xyloglucan endotransglycosylase action in Arabidopsis roots.

Root hairs are formed by two separate processes: initiation and subsequent tip growth. Root hair initiation is always accompanied by a highly localized increase in xyloglucan endotransglycosylase (XET) action at the site of future bulge formation, where the trichoblast locally loosens its cell wall. This suggests an important role of XET in the first stages of root hair initiation. The tip of growing root hairs is not marked by localized high XET action. Experiments in which root hair initiation was modulated and observations on root hair mutants support this view. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid shifts both root hair initiation and the local increase in XET action toward the root tip. On the other hand, roots treated with the ethylene inhibitor aminoethoxyvinyl-glycine, as well as roots of mutants affected in root hair initiation (rhl1, rhd6-1, and axr2-1) revealed no localized increases of XET action at all and consequently did not initiate root hairs. Disruption of actin and microtubules did not prevent the localized increase in XET action. Also, the temporal and spatial pattern of action as the specific pH dependence suggest that different isoforms of XET act in different processes of root development.

Density-labelling of cell wall polysaccharides in cultured rose cells: comparison of incorporation of 2H and 13C from exogenous glucose.

Labelling with stable isotopes has under-exploited potential for studies of polysaccharide endotransglycosylation in vivo. Ideally, the labelled polysaccharides should have the highest possible buoyant density. Although [13C6]glucose has previously been used as a precursor, it was unclear whether 2H would be efficiently incorporated from [2H]glucose or lost as D2O. Rose (Rosa sp.) cell-suspension cultures efficiently incorporated 13C from D-[13C6,2H7]glucose into wall polysaccharides with negligible dilution from atmospheric 12CO2. Also, approximately 70% of the 2H atoms in D-[13C6,2H7]glucose were retained during polysaccharide biosynthesis. This shows that relatively few cycles of intermediary metabolism leading to the release of D2O occurred before sugar residues were incorporated into wall polysaccharides. In agreement with these observations, isopycnic centrifugation in caesium trifluoroacetate gradients showed that the hydrated buoyant density of xyloglucan synthesised by rose cells growing on [13C6,2H7]glucose and [13C6]glucose was 3.7 and 2.6% higher, respectively, than in isotopically non-labelled cultures. Thus, [13C,2H]glucose-feeding enabled a 42% better resolution of 'heavy' from 'light' xyloglucan than [13C]glucose-feeding.

Characteristics of xyloglucan after attack by hydroxyl radicals.

It has been proposed that plant cell-wall polysaccharides are subject in vivo to non-enzymic scission mediated by hydroxyl radicals (-*OH). In the present study, xyloglucan was subjected in vitro to partial, non-enzymic scission by treatment with ascorbate plus H(2)O(2), which together generate -*OH. The partially degraded xyloglucan appeared to contain ester bonds within the backbone, as indicated by an irreversible decrease in viscosity upon alkaline hydrolysis. Aldehyde and/or ketone groups were also introduced into the polysaccharide by -*OH-attack, as indicated by staining with aniline hydrogen-phthalate and by reaction with NaB(3)H(4). The introduction of ester and oxo groups supports the proposed sequence of reactions: (a) -*OH-mediated H-abstraction to produce a carbon-centred carbohydrate radical; (b) reaction of the latter with O(2); and (c) elimination of a hydroperoxyl radical (HO(2)*-). When the partially degraded xyloglucan was reduced with NaB(3)H(4) followed by acid hydrolysis, several 3H-aldoses were detected ([3H]galactose, [3H]xylose, [3H]glucose, [3H]ribose and probably [3H]mannose), in addition to unidentified 3H-products (probably including anhydroaldoses). 3H-Alditols were undetectable, showing that few or no conventional reducing termini were introduced. Digestion of the NaB(3)H(4)-reduced, partially degraded xyloglucan with Driselase released 25 times more [3H]Xyl-alpha-(1-->6)-Glc than Xyl-alpha-(1-->6)-[3H]Glc, suggesting that the xylose side-chains of the xyloglucan had been more heavily attacked by -*OH than the glucose residues of the backbone. The radioactive xyloglucan was readily digested by cellulase, yielding 3H-products in the hepta- to nonasaccharide range. A fingerprinting strategy for identifying -*OH-attacked xyloglucan in plant cell walls is proposed.

Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit.

Hydroxyl radicals (*OH) may cause non-enzymic scission of polysaccharides in vivo, e.g. in plant cell walls and mammalian connective tissues. To provide a method for detecting the action of endogenous *OH in vivo, we investigated the products formed when polysaccharides were treated with *OH (generated in situ by ascorbate-H(2)O(2)-Cu(2+) mixtures) followed by NaB(3)H(4). Treatment with *OH increased the number of NaB(3)H(4)-reacting groups present in citrus pectin, homogalacturonan and tamarind xyloglucan. This increase is attributed partly to the formation of glycosulose and glycosulosuronic acid residues, which are then reduced back to the original (but radioactive) sugar residues and their epimers by NaB(3)H(4). The glycosulose and glycosulosuronic acid residues were stable for >16 h at 20 degrees C in ethanol or buffer (pH 4.7), but were destroyed in alkali. Driselase-digestion of the radiolabelled polysaccharides yielded characteristic patterns of (3)H-products, which included galactose and galacturonate from pectin, and isoprimeverose, galactose, glucose and arabinose from xyloglucan. Pectin yielded at least eight (3)H-labelled anionic products, separable by electrophoresis at pH 3.5. The patterns of radioactive products form useful 'fingerprints' by which *OH-attacked polysaccharides may be recognized. Applied to the cell walls of ripening pear (Pyrus communis) fruit, the method gave evidence for progressive *OH radical attack on polysaccharides during the softening process.

3-O-methyl-D-galactose residues in lycophyte primary cell walls.

Acid hydrolysis of cell wall-rich material from young leaves of the lycophyte Selaginella apoda (L.) Spring yielded substantial amounts of 3-O-methyl-D-galactose (1) in addition to the usual major monosaccharides (glucose, galactose, arabinose, xylose and galacturonic acid). The yield of 1 approximately equalled that of galacturonic acid. Compound 1 was identified as 3-O-methylgalactose by its 1H and 13C NMR spectra, and shown to be the D-enantiomer by its susceptibility to D-galactose oxidase. Compound 1 was detected in acid hydrolysates of the alcohol-insoluble residues from young leaves of all lycophytes tested, both homosporous (Lycopodium, Huperzia and Diphasiastrum) and heterosporous (Selaginella). It was not detectable in the charophyte green algae Coleochaete scutata, Chara coralina or Klebsormidium flaccidum, any bryophytes [a hornwort (Anthoceros), four liverworts and three mosses], or any euphyllophytes [a psilopsid (Psilotum), a horsetail (Equisetum), eusporangiate and leptosporangiate ferns, the gymnosperm Gnetum, and diverse angiosperms]. A high content of 1 is thus an autapomorphy of the lycophytes.

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.

ESR study of the non-enzymic scission of xyloglucan by an ascorbate-H2O2-copper system: the involvement of the hydroxyl radical and the degradation of ascorbate.

Xyloglucan is degraded by a mixture of copper(II), hydrogen peroxide and ascorbate. In the presence of ascorbate and/or hydrogen peroxide, copper(II) species were rapidly reduced to copper(I), which react with hydrogen peroxide. Spin-trapping experiments showed that hydroxyl radicals formed and attacked xyloglucan causing its degradation. The formation of a carbon-centred ascorbyl (C-ascorbyl) radical and its degradation with the formation of oxalate, was also caused by hydroxyl radicals. As a consequence, the features of the bis(oxalate) copper(II) complex clearly appeared in the frozen solution ESR spectra. The formation of carbon-centred radicals on the xyloglucan is the trigger for a series of possible molecular rearrangements which led to its oxidative scission.

Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically.

To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) along the donor substrate chain, we incubated the enzymes with tamarind (Tamarindus indica) xyloglucan (donor substrate; approximately 205 kDa; 21 microM) plus the nonasaccharide [(3)H]XLLGol (Gal(2).Xyl(3).Glc(3). [(3)H]glucitol; acceptor substrate; 0.6 microM). After short incubation times, to minimize multiple cleavages, the size of the (3)H-labelled transglycosylation products (determined by gel-permeation chromatography) indicated the positions of the cleavage sites relative to the non-reducing terminus of the donor. There was very little difference between the size profiles of the products formed by any of ten XETs tested [one native XET purified from cauliflower (Brassica oleracea) florets, four native XET isoenzymes purified from etiolated mung-bean (Phaseolus aureus) shoots, native XETs purified from lentil (Lens culinaris) and nasturtium (Tropaeolum majus) seeds, and three insect-cell-produced thale-cress (Arabidopsis thaliana) XETs (EXGT, TCH4 and MERI-5)]. All such product profiles showed a good fit to a model in which the enzyme chooses its donor substrate independently of size and attacks it, once only, at a randomly selected cleavage site. The results therefore do not support the hypothesis that different XET isoenzymes are adapted to produce longer or shorter products such as might favour either the efficient integration of new xyloglucan into the cell wall or the re-structuring of old xyloglucan within an expanding wall.

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures.

Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5-20 min, the CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse-chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1-3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised < 1 min after their attachment to polysaccharides. For at least the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at approximately 7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides < 1 min after the biosynthesis of the latter, and > 10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET).

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.

Chitinases from Vibrio: activity screening and purification of chiA from Vibrio carchariae.

Fourteen species of Vibrio were screened for chitin-induced chitinase activity in culture medium. V. carchariae, V. alginolyticus 283 and V. campbellii showed high levels of activity. Screening on agar plates containing swollen chitin showed high levels of chitinase activity by the same three species, and also by V. fischeri and V. alginolyticus 284. An affinity purification procedure was developed for the chitinase from V. carchariae. The purified chitinase was active as a monomer with M(r) 63,000-66,000, and displayed activity toward polymeric chitin from acetylated chitosan or from crab shells. N-terminal sequence analysis and immunological cross-reactivity confirmed that the enzyme belongs to the group A/chiA family of bacterial chitinases.

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

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.

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.

Properties of herbage in relation to equine dysautonomia: biochemical composition and antioxidant and prooxidant actions.

To investigate the etiology of equine dysautonomia (ED), a degenerative polyneuropathy affecting grazing horses, the biochemical composition and antioxidant/prooxidant activities of aqueous extracts of plants collected from ED pastures were determined. Plants collected immediately after an outbreak of ED had reduced antioxidant and weak prooxidant activities when compared with control plants (plants collected from ED pastures out of ED season and control plants from ED pastures that were grown under favorable conditions). ED plants also had significantly increased concentrations of fructose and low molecular weight phenolic compounds, significantly more of one amino acid zone (probably valine), significantly less tartaric acid, and a nonsignificant decrease in ascorbic acid content when compared with control plants from ED pastures that were grown under favorable conditions. These findings suggest that ED plants may be under oxidative stress, possibly due to chilling, drought, or fungal colonization. However, experimental drought and chilling of plants did not reproduce the biochemical alterations identified in ED plants. It is possible that the altered biochemical content of ingested plants may contribute, directly or indirectly, to the development of ED in grazing horses.

Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex.

We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)2SO4, were incubated with tamarind xyloglucan (approximately 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses >500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximately 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE.

Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals.

Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of l-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1 mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10-25-min delay. Catalase prevented the scission, whereas added H2O2 (0.1-10 mM) increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above approx. 5 microM. Dehydroascorbate was much less effective. Added Cu2+ (>0.3 microM) also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu (2.8 mg.kg-1) present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers; their effectiveness was proportional to their rate constants for reaction with hydroxyl radicals (.OH). It is proposed that ascorbate non-enzymically reduces O2 to H2O2, and Cu2+ to Cu+, and that H2O2 and Cu+ react to form .OH, which causes oxidative scission of polysaccharide chains. Evidence is reviewed to suggest that, in the wall of a living plant cell, Cu+ and H2O2 are formed by reactions involving ascorbate and its products, dehydroascorbate and oxalate. Systems may thus be in place to produce apoplastic .OH radicals in vivo. Although .OH radicals are often regarded as detrimental, they are so short-lived that they could act as site-specific oxidants targeted to play a useful role in loosening the cell wall, e.g. during cell expansion, fruit ripening and organ abscission.

Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl-enzyme intermediate.

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xyloglucan moiety for cellulose. The XET-xyloglucan complex was stable in water, 6 M urea and acidic and alkaline buffers (pH 2.5-9.5), but readily decomposed by transferring its glycosyl moiety to xyloglucan-derived oligosaccharides or by incubation with the strong nucleophile imidazole at pH 3.8-9.6. These results strongly support the assumption that XET forms a relatively stable covalently linked glycosyl-enzyme intermediate.

Pulcherosine, an oxidatively coupled trimer of tyrosine in plant cell walls: its role in cross-link formation.

An oxidatively coupled trimer of tyrosine has been isolated from hydrolysates of primary cell walls of a tomato cell culture. UV-absorption, fluorescence and 1H NMR spectra showed that the trimer was pulcherosine, composed of isodityrosine and tyrosine oxidatively coupled via a biphenyl linkage such that the aromatic core is 2,2'-dihydroxy-3-phenoxybiphenyl. Pulcherosine could act as an intermediate in the conversion of isodityrosine to the tetramer, di-isodityrosine. Steric considerations show that the three tyrosine units of pulcherosine could not be near-neighbour residues within a single polypeptide chain. Pulcherosine therefore forms inter-polypeptide cross-links and/or wide intra-polypeptide loops.