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SUMMARY: Cross‐linking of the cell‐wall pectin domain rhamnogalacturonan‐II (RG‐II) via boron bridges between apiose residues is essential for normal plant growth and development, but little is known about its mechanism or reversibility. We characterized the making and breaking of boron bridges in vivo and in vitro at 'apoplastic' pH. RG‐II (13–26 μm) was incubated in living Rosa cell cultures and cell‐free media with and without 1.2 mm H3BO3 and cationic chaperones (Ca2+, Pb2+, polyhistidine, or arabinogalactan‐protein oligopeptides). The cross‐linking status of RG‐II was monitored electrophoretically. Dimeric RG‐II was stable at pH 2.0–7.0 in vivo and in vitro. In‐vitro dimerization required a 'catalytic' cation at all pHs tested (1.75–7.0); thus, merely neutralizing the negative charge of RG‐II (at pH 1.75) does not enable boron bridging. Pb2+ (20–2500 μm) was highly effective at pH 1.75–4.0, but not 4.75–7.0. Cationic peptides were effective at approximately 1–30 μm; higher concentrations caused less dimerization, probably because two RG‐IIs then rarely bonded to the same peptide molecule. Peptides were ineffective at pH 1.75, their pH optimum being 2.5–4.75. d‐Apiose (>40 mm) blocked RG‐II dimerization in vitro, but did not cleave existing boron bridges. Rosa cells did not take up d‐[U‐14C]apiose; therefore, exogenous apiose would block only apoplastic RG‐II dimerization in vivo. In conclusion, apoplastic pH neither broke boron bridges nor prevented their formation. Thus boron‐starved cells cannot salvage boron from RG‐II, and 'acid growth' is not achieved by pH‐dependent monomerization of RG‐II. Divalent metals and cationic peptides catalyse RG‐II dimerization via co‐ordinate and ionic bonding respectively (possible and impossible, respectively, at pH 1.75). Exogenous apiose may be useful to distinguish intra‐ and extra‐protoplasmic dimerization. Significance Statement: This study demonstrates the nature of the interaction between the cell‐wall pectic domain rhamnogalacturonan‐II (RG‐II) and cationic chaperones necessary for efficient RG‐II dimerization via borate diester bonds at acidic pH, resembling the acid growth conditions in the cell wall. Boron bridging is irreversible at apoplastic pH, so boron‐starved cells cannot salvage boron from RG‐II, and wall loosening in 'acid growth' is not achieved by pH‐dependent modification of RG‐II cross‐linking. [ABSTRACT FROM AUTHOR]

SUMMARY: Transglycanases remodel cell‐wall polymers, having a critical impact on many physiological processes. Unlike xyloglucan endotransglucosylase (XET) activity, widely studied in land plants, very little is known about charophyte wall‐modifying enzymes – information that would promote our understanding of the 'primordial' wall, revealing how the wall matrix is remodelled in the closest living algal relatives of land plants, and what changed during terrestrialisation. We conducted various in‐vitro assays for wall‐remodelling transglycosylases, monitoring either (a) polysaccharide‐to‐[3H]oligosaccharide transglycosylation or (b) non‐radioactive oligosaccharide‐to‐oligosaccharide transglycosylation. We screened a wide collection of enzyme extracts from charophytes (and early‐diverging land plants for comparison) and discovered several homo‐ and hetero‐transglycanase activities. In contrast to most land plants, charophytes possess high trans‐β‐1,4‐mannanase activity, suggesting that land plants' algal ancestors prioritised mannan remodelling. Trans‐β‐1,4‐xylanase activity was also found, most abundantly in Chara, Nitella and Klebsormidium. Exo‐acting transglycosidase activities (trans‐β‐1,4‐xylosidase and trans‐β‐1,4‐mannosidase) were also detected. In addition, charophytes exhibited homo‐ and hetero‐trans‐β‐glucanase activities (XET, mixed‐linkage glucan [MLG]:xyloglucan endotransglucosylase and cellulose:xyloglucan endotransglucosylase) despite the paucity or lack of land‐plant‐like xyloglucan and MLG as potential donor substrates in their cell walls. However, trans‐α‐xylosidase activity (which remodels xyloglucan in angiosperms) was absent in charophytes and early‐diverging land plants. Transglycanase action was also found in situ, acting on endogenous algal polysaccharides as donor substrates and fluorescent xyloglucan oligosaccharides as acceptor substrates. We conclude that trans‐β‐mannanase and trans‐β‐xylanase activities are present and thus may play key roles in charophyte walls (most of which possess little or no xyloglucan and MLG, but often contain abundant β‐mannans and β‐xylans), comparable to the roles of XET in xyloglucan‐rich land plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Certain transglucanases can covalently graft cellulose and mixed‐linkage β‐glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero‐transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero‐transglucosylation, we studied Equisetum fluviatile hetero‐trans‐β‐glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo‐transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia‐produced EfHTG was up to approximately 300% increased by addition of methanol‐boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol‐stable factor is proposed to be expansin‐like, a suggestion supported by observations of pH dependence. Screening numerous low‐molecular‐weight compounds for hetero‐transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero‐transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero‐transglucosylation in planta by identifying factors that govern this reaction. Significance Statement: The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re‐structuring the cell wall via hetero‐transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin‐enriched preparation was added. Hetero‐transglycosylation was inhibited by cellobiose – a potential tool for exploring HTG's biological functions. Interestingly, cellobiose retarded Equisetum stem elongation, suggesting a role for HTG in growth. [ABSTRACT FROM AUTHOR]

Summary: Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall‐modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed‐linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components. Significance Statement: This study suggests that xyloglucan endotransglucosylase/hydrolases from Fragaria vesca, namely FvXTH9 and FvXTH6, promote strawberry fruit ripening through involvement in the modification of cell wall components. Overexpression of FvXTH9 and FvXTH6 in Fragaria × ananassa fruits resulted in accelerated fruit softening. [ABSTRACT FROM AUTHOR]

Summary: In the plant apoplast, ascorbate is oxidised, via dehydroascorbic acid, to O‐oxalyl esters [oxalyl‐ l‐threonate (OxT) and cyclic oxalyl‐ l‐threonate (cOxT)]. We tested whether OxT and cOxT can donate the oxalyl group in transacylation reactions to form oxalyl‐polysaccharides, potentially modifying the cell wall. [oxalyl‐14C]OxT was incubated with living spinach (Spinacia oleracea) and Arabidopsis cell‐suspension cultures in the presence or absence of proposed acceptor substrates (carbohydrates). In addition, [14C]OxT and [14C]cOxT were incubated in vitro with cell‐wall enzyme preparations plus proposed acceptor substrates. Radioactive products were monitored electrophoretically. Oxalyltransferase activity was detected. Living cells incorporated oxalate groups from OxT into cell‐wall polymers via ester bonds. When sugars were added, [14C]oxalyl‐sugars were formed, in competition with OxT hydrolysis. Preferred acceptor substrates were carbohydrates possessing primary alcohols e.g. glucose. A model transacylation product, [14C]oxalyl‐glucose, was relatively stable in vivo (half‐life >24 h), whereas [14C]OxT underwent rapid turnover (half‐life ~6 h). Ionically wall‐bound enzymes catalysed similar transacylation reactions in vitro with OxT or cOxT as oxalyl donor substrates and any of a range of sugars or hemicelluloses as acceptor substrates. Glucosamine was O‐oxalylated, not N‐oxalylated. We conclude that plants possess apoplastic acyltransferase (oxalyltransferase) activity that transfers oxalyl groups from ascorbate catabolites to carbohydrates, forming relatively long‐lived O‐oxalyl‐carbohydrates. The findings increase the range of known metabolites whose accumulation in vivo indicates vitamin C catabolism. Possible signalling roles of the resulting oxalyl‐sugars can now be investigated, as can the potential ability of polysaccharide oxalylation to modify the wall's physical properties. [ABSTRACT FROM AUTHOR]

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non-reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [14C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall-rich) leaf material. No [14C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under-expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation. [ABSTRACT FROM AUTHOR]

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage β-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-β-glucanase ( HTG), a transglycanase that targets cellulose, in horsetails ( Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage β-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage β-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass. [ABSTRACT FROM AUTHOR]

Boron (B) is essential for plant cell-wall structure and membrane functions. Compared with its role in cross-linking the pectic domain rhamnogalacturonan II ( RG- II), little information is known about the biological role of B in membranes. Here, we investigated the involvement of glycosylinositol phosphorylceramides ( GIPCs), major components of lipid rafts, in the membrane requirement for B. Using thin-layer chromatography and mass spectrometry, we first characterized GIPCs from Rosa cell culture. The major GIPC has one hexose residue, one hexuronic acid residue, inositol phosphate, and a ceramide moiety with a C18 trihydroxylated mono-unsaturated long-chain base and a C24 monohydroxylated saturated fatty acid. Disrupting B bridging (by B starvation in vivo or by treatment with cold dilute HCl or with excess borate in vitro) enhanced the GIPCs' extractability. As RG- II is the main B-binding site in plants, we investigated whether it could form a B-centred complex with GIPCs. Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG- II, suggesting formation of a GIPC-B- RG- II complex. Last, using polyacrylamide gel electrophoresis, we showed that added GIPCs facilitate RG- II dimerization in vitro. We conclude that B plays a structural role in the plasma membrane. The disruption of membrane components by high borate may account for the phytotoxicity of excess B. Moreover, the in-vitro formation of a GIPC-B- RG- II complex gives the first molecular explanation of the wall-membrane attachment sites observed in vivo. Finally, our results suggest a role for GIPCs in the RG- II dimerization process. [ABSTRACT FROM AUTHOR]

The cell-wall pectic domain rhamnogalacturonan- II ( RG- II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis ( PAGE) to separate monomeric from dimeric (boron-bridged) RG- II, we confirmed that Pb2+ promotes H3 BO3-dependent dimerisation in vitro. H3 BO3 concentrations as high as 50 m m did not prevent cross-linking. For in-vivo experiments, we successfully cultured 'Paul's Scarlet' rose ( Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG- II domains but no detectable dimers. Thus pectins containing RG- II domains can be held in the wall other than via boron bridges. Re-addition of H3 BO3 to 3.3 μ m triggered a gradual appearance of RG- II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG- II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG- II dimers. We conclude that RG- II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [3H] RG- II was neither dimerised in the medium nor cross-linked to existing wall-associated RG- II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG- II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur. [ABSTRACT FROM AUTHOR]

Cell-wall components are hydrolysed by numerous plant glycosidase and glycanase activities. We investigated whether plant enzymes also modify xyloglucan structures by transglycosidase activities. Diverse angiosperm extracts exhibited transglycosidase activities that progressively transferred single sugar residues between xyloglucan heptasaccharide (XXXG or its reduced form, XXXGol) molecules, at 16 μ m and above, creating octa- to decasaccharides plus smaller products. We measured remarkably high transglycosylation:hydrolysis ratios under optimized conditions. To identify the transferred monosaccharide(s), we devised a dual-labelling strategy in which a neutral radiolabelled oligosaccharide (donor substrate) reacted with an amino-labelled non-radioactive oligosaccharide (acceptor substrate), generating radioactive cationic products. For example, 37 μ m [ Xyl-3H]XXXG plus 1 m m XXLG-NH2 generated 3H-labelled cations, demonstrating xylosyl transfer, which exceeded xylosyl hydrolysis 1.6- to 7.3-fold, implying the presence of enzymes that favour transglycosylation. The transferred xylose residues remained α-linked but were relatively resistant to hydrolysis by plant enzymes. Driselase digestion of the products released a trisaccharide (α-[3H]xylosyl-isoprimeverose), indicating that a new xyloglucan repeat unit had been formed. In similar assays, [ Gal-3H]XXLG and [ Gal-3H]XLLG (but not [ Fuc-3H]XXFG) yielded radioactive cations. Thus plants exhibit trans-α-xylosidase and trans-β-galactosidase (but not trans-α-fucosidase) activities that graft sugar residues from one xyloglucan oligosaccharide to another. Reconstructing xyloglucan oligosaccharides in this way may alter oligosaccharin activities or increase their longevity in vivo. Trans-α-xylosidase activity also transferred xylose residues from xyloglucan oligosaccharides to long-chain hemicelluloses (xyloglucan, water-soluble cellulose acetate, mixed-linkage β-glucan, glucomannan and arabinoxylan). With xyloglucan as acceptor substrate, such an activity potentially affects the polysaccharide's suitability as a substrate for xyloglucan endotransglucosylase action and thereby modulates cell expansion. We conclude that certain proteins annotated as glycosidases can function as transglycosidases. [ABSTRACT FROM AUTHOR]

Summary Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides 'modelling' selected cell-wall polysaccharides. Based on reaction products, we successfully distinguished exo- and endo-hydrolases and found high taxonomic variation in all hydrolases screened: β- d-xylosidase, endo-(1→4)-β- d-xylanase, β- d-mannosidase, endo-(1→4)-β- d-mannanase, α- d-xylosidase, β- d-galactosidase, α- l-arabinosidase and α- l-fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during β-xylosidase assays on (1→4)-β- d-xylohexaose (Xyl6), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl5 and Xyl7, indicating trans-β-xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl9 often exceeded that of Xyl7-8, indicating that β-xylanase was accompanied by an endotransglycosylase activity, here called trans-β-xylanase, catalysing the reaction 2Xyl6→ Xyl3 + Xyl9. Similar evidence also revealed trans-α-xylosidase, trans-α-arabinosidase and trans-α-arabinanase activities acting on xyloglucan oligosaccharides and (1→5)-α- l-arabino-oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re-structuring of the wall matrix. [ABSTRACT FROM AUTHOR]

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross-link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross-linking in vivo are unclear. Non-lignifying maize ( Zea mays L.) cell cultures were incubated withl-[1-3H]arabinose or ( E)-[U-14C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl-[3H]arabinoxylans. The cross-linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL-2B, peaked suddenly and transiently, typically at ∼9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H2O2 and/or inhibitors. De-esterified arabinoxylans failed to cross-link, supporting a role for the feruloyl ester groups. The cross-links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p-hydroxybenzyl–sugar ether bonds. Cross-linking of [14C]feruloyl-arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H2O2. During cross-linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin-layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali-stable (ether-like) bonds. Thus, feruloyl esters participate in polysaccharide cross-linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p-hydroxybenzyl–polysaccharide ether bonds are formed via quinone-methide intermediates. [ABSTRACT FROM AUTHOR]

Mixed-linkage (1→3,1→4)-β-d-glucan (MLG), a hemicellulose long thought to be confined to certain Poales, was recently also found in Equisetum; xyloglucan occurs in all land plants. We now report that Equisetum possesses MLG:xyloglucan endotransglucosylase (MXE), which is a unique enzyme that grafts MLG to xyloglucan oligosaccharides (e.g. the heptasaccharide XXXGol). MXE occurs in all Equisetum species tested ( Equisetum arvense, Equisetum fluviatile, Equisetum hyemale, Equisetum scirpoides, Equisetum telmateia and Equisetum variegatum), sometimes exceeding xyloglucan endotransglucosylase (XET) activity. Charophytic algae, especially Coleochaete, also possess MXE, which may therefore have been a primordial feature of plant cell walls. However, MXE was negligible in XET-rich extracts from grasses, dicotyledons, ferns, Selaginella and bryophytes. This and the following four additional observations indicate that MXE activity is not the result of a conventional xyloglucan endotransglucosylase/hydrolase (XTH): (i) XET, but not MXE, activity correlates with the reaction rate on water-soluble cellulose acetate, hydroxyethylcellulose and carboxymethylcellulose, (ii) MXE and XET activities peak in old and young Equisetum stems, respectively, (iii) MXE has a higher affinity for XXXGol ( Km ∼ 4 μM) than any known XTH, (iv) MXE and XET activities differ in their oligosaccharide acceptor-substrate preferences. High-molecular-weight ( Mr) xyloglucan strongly competes with [3H]XXXGol as the acceptor-substrate of MXE, whereas MLG oligosaccharides are poor acceptor-substrates. Thus, MLG-to-xyloglucan grafting appears to be the favoured activity of MXE. In conclusion, Equisetum has evolved MLG plus MXE, potentially a unique cell wall remodelling mechanism. The prominence of MXE in mature stems suggests a strengthening/repairing role. We propose that cereals, which possess MLG but lack MXE, might be engineered to express this Equisetum enzyme, thereby enhancing the crop mechanical properties. [ABSTRACT FROM AUTHOR]

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5′-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially ‘competing’ pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces ofd-[1-3H]galactose and a 14C-labelled hexose (e.g.d-[U-14C]fructose) in the presence of an approximately 104-fold excess of non-radioactive carbon source. Six interconvertible ‘core intermediates’, galactose 1-phosphate ↔ UDP-galactose ↔ UDP-glucose ↔ glucose 1-phosphate ↔ glucose 6-phosphate ↔ fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5′-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]