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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.

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.

Transglycanases (endotransglycosylases) cleave a polysaccharide (donor-substrate) in mid-chain, and then transfer a portion onto another poly- or oligosaccharide (acceptor-substrate). Such enzymes contribute to plant cell-wall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated (3)H-polysaccharides from unreacted (3)H-oligosaccharides--the former immobilized (on filter-paper, silica-gel or glass-fiber), the latter eluted. On filter-paper, certain polysaccharides [e.g. (1→3, 1→4)-β-D-glucans] remained satisfactorily adsorbed when water-washed; others (e.g. pectins) were partially lost. Many oligosaccharides (e.g. arabinan-, galactan-, xyloglucan-based) were successfully eluted in appropriate solvents, but others (e.g. [(3)H]xylohexaitol, [(3)H]mannohexaitol [(3)H]cellohexaitol) remained immobile. On silica-gel, all (3)H-oligosaccharides left an immobile 'ghost' spot (contaminating any (3)H-polysaccharides), which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glass-fiber (GF), onto which the reaction-mixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of (3)H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris trans-β-mannanase. In conclusion, our novel GF-blotting technique efficiently frees transglycanase-generated (3)H-polysaccharides from unreacted (3)H-oligosaccharides, enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor- and acceptor-substrates.

Cell-suspension cultures (Zea mays L., Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile (DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to define the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose, and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into (hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular trafficking of polymers, especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log-phase cultures, habituation increased the proportion of 3H-hemicelluloses ([3H]xylans and [3H]xyloglucan) sloughed into the medium. These findings could be related to the cell walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also reflect the habituated cells' reduced capacity to integrate arabinoxylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.

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.

Summary Among land-plant hemicelluloses, xyloglucan is ubiquitous, whereas mixed-linkage (1→3),(1→4)-β-d-glucan (MLG) is confined to the Poales (e.g. cereals) and Equisetales (horsetails). The enzyme MLG:xyloglucan endotransglucosylase (MXE) grafts MLG to xyloglucan. In Equisetum, MXE often exceeds extractable xyloglucan endotransglucosylase (XET) activity; curiously, cereals lack extractable MXE. We investigated whether barley possesses inextractable MXE. Grafting of endogenous MLG or xyloglucan onto exogenous [3H]xyloglucan oligosaccharides in vivo indicated MXE and XET action, respectively. Extractable MXE and XET activities were assayed in vitro. MXE and XET actions were both detectable in living Equisetum fluviatile shoots, the MXE : XET ratio increasing with age. However, only XET action was observed in barley coleoptiles, leaves and roots (which all contained MLG) and in E. fluviatile intercalary meristems and callus (which lacked MLG). In E. fluviatile, extractable MXE activity was high in mature shoots, but extremely low in callus and young shoots; in E. arvense strobili, it was undetectable. Barley possesses neither extractable nor inextractable MXE, despite containing both of its substrates and high XET activity. As the Poales are xyloglucan-poor, the role of their abundant endotransglucosylases remains enigmatic. The distribution of MXE action and activity within Equisetum suggests a strengthening role in ageing tissues.

Summary 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 μm 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 μm [Xyl-3H]XXXG plus 1 mm 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.

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.

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 with l-[1-(3)H]arabinose or (E)-[U-(14)C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl-[(3)H]arabinoxylans. The cross-linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL-2B, peaked suddenly and transiently, typically at approximately 9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H(2)O(2) 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 [(14)C]feruloyl-arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H(2)O(2). 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.

Summary • Recent studies demonstrating an in situ formation of methane (CH4) within foliage and separate observations that soil-derived CH4 can be released from the stems of trees have continued the debate about the role of vegetation in CH4 emissions to the atmosphere. Here, a study of the role of ultraviolet (UV) radiation in the formation of CH4 and other trace gases from plant pectins in vitro and from leaves of tobacco (Nicotiana tabacum) in planta is reported. • Plant pectins were investigated for CH4 production under UV irradiation before and after de-methylesterification and with and without the singlet oxygen scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO). Leaves of tobacco were also investigated under UV irradiation and following leaf infiltration with the singlet oxygen generator rose bengal or the bacterial pathogen Pseudomonas syringae. • Results demonstrated production of CH4, ethane and ethylene from pectins and from tobacco leaves following all treatments, that methyl-ester groups of pectin are a source of CH4, and that reactive oxygen species (ROS) arising from environmental stresses have a potential role in mechanisms of CH4 formation. • Rates of CH4 production were lower than those previously reported for intact plants in sunlight but the results clearly show that foliage can emit CH4 under aerobic conditions.

Mixed-linkage (1--3,1--4)-beta-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 (K(m) approximately 4 microM) than any known XTH, (iv) MXE and XET activities differ in their oligosaccharide acceptor-substrate preferences. High-molecular-weight (M(r)) xyloglucan strongly competes with [(3)H]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.

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 of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-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.

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous D-[U- 14 C]glucose, D-[U- 14 C]mannose or D-[2- 3 H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose-, arabinose- and mannose-containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by p-glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose-rich poly- and oligosaccharides.

Summary • Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. • Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of 3H2O and [14C]mannitol from a ‘bed’ of cultured cells. Osmotic potential was measured by depression of freezing point. • OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID50 of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells’ ability to acidify the apoplast (medium), which may be necessary for ‘acid growth’. • As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.

Numerous examples have been presented of enzyme activities, assayed in vitro, that appear relevant to the synthesis of structural polysaccharides, and to their assembly and subsequent degradation in the primary cell walls (PCWs) of higher plants. The accumulation of the corresponding mRNAs, and of the (immunologically recognized) proteins, has often also (or instead) been reported. However, the presence of these mRNAs, antigens and enzymic activities has rarely been shown to correspond to enzyme action in the living plant cell. In some cases, apparent enzymic action is observed in vivo for which no enzyme activity can be detected in in-vitro assays; the converse also occurs. Methods are reviewed by which reactions involving structural wall polysaccharides can be tracked in vivo. Special attention is given to xyloglucan endotransglucosylase (XET), one of the two enzymic activities exhibited in vitro by xyloglucan endotransglucosylase/hydrolase (XTH) proteins, because of its probable importance in the construction and restructuring of the PCW's major hemicellulose. Attention is also given to the possibility that some reactions observed in the PCW in vivo are not directly enzymic, possibly involving the action of hydroxyl radicals. It is concluded that some proposed wall enzymes, for example XTHs, do act in vivo, but that for other enzymes this is not proven. Contents I. Primary cell walls: composition, deposition and roles 642 II. Reactions that have been proposed to occur in primary cell walls 645 III. Tracking the careers of wall components in vivo: evidence for action of enzymes in the walls of living plant cells 656 IV. Evidence for the occurrence of nonenzymic polymer scission in vivo? 666 VI. Conclusion 667 References 667.

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.

The aim of the work was to study the binding, degradation and metabolism of dietary condensed tannins in the gastrointestinal tract of an omnivore. Young pods of carob (Ceratonia siliqua L) were radiolabelled by in vivo feeding of 14 CO 2 , trans-[U-14C]cinnamate or L-[U-14C]phenylalanine. [ 14 C]Proanthocyanidins (condensed tannins) were extracted with acetone/water (3:1 v/v), isolated on Sephadex LH-20 and fed to rats by gavage. After 4 and 18h, 90-94% of the gavaged 14 C was in the gut contents and/or faeces. Much of the gavaged 14 C (57%), predominantly that originally in tannins of high degree of polymerisation (DP), became insolubilised, mainly in the form of protein-tannin complexes. Some of the [ 14 C]tannins that remained soluble decreased in DP, especially in the small intestine and caecum. A further fraction (12% of the 14 C gavaged) underwent chemical modifications in the gut to form soluble, non-tannin compounds. Small proportions of the 14 C were found in the liver (1.0-1.5%), urine (1-2%) and 14 CO 2 (1-2%). We conclude that proanthocyanidins are not inert within the gut but undergo various modifications which may affect the nutrition of the animal. Les gousses de caroube (Ceratonia siliqua L.) sont utilisees d'une part, en industrie alimentaire comme un substitut du chocolat et d'autre part dans la preparation de produit antidiarrheiques et antiemetiques ainsi que pour l'alimentation animale. La gousse contient des teneurs importantes en tanins condenses (proanthocyanidines) hautement polymerises. La capacite de ces composes a former des complexes avec les proteines fait qu'on les classe dans les facteurs antinutritionnels. Cependant, des etudes recentes ont montre que les anthocyanidines ont aussi des proprietes cardioprotectrices. L'objet de cette etude est de determiner le comportement d'anthocyanidines de caroube dans le tractus intestinal du rat, leur absorption et leur metabolisme. Les resultats montrent qu'elles subissent des modifications variees qui peuvent affecter la nutrition de l'animal.

A novel dot-blot method is described for the rapid, semi-quantitative assay of xyloglucan endotransglycosylase (XET) activity. Test paper was prepared by impregnating Whatman No. 1 filter paper with xyloglucan (2.5 g m−2 paper) plus a sulphorhodamine conjugate of the xyloglucan oligosaccharide, XLLG (1 µmol m−2). Spots of putative enzyme solution were applied to the test paper, which was incubated under humid conditions. XET-catalysed transglycosylation produced a xyloglucan-sulphorhodamine conjugate. The test paper was washed to remove unreacted oligosaccharide, leaving fluorescent spots of the xyloglucan-sulphorhodamine reaction product hydrogen-bonded to the paper. The assay is suitable for testing crude plant extracts (including Arabidopsis stems squashed directly on to the test paper). The test paper is also suitable for tissue-printing of plant specimens and for preparation of zymograms after non-denaturing gel electrophoresis. Tissue prints indicated that extractable XET activity tends to be concentrated in the outer tissues and sometimes in or near the vascular bundles. The method reported can be quantified by fluorimetry and can potentially be extended to the assay of other transglycosylases (including polysaccharide synthases, fructosyl-transferase and dextransucrase) and hydrolases.

It has been shown that a common side-chain of grass cell wall arabinoxylans is 2-O-β-D-xylopyranosyl-(5-O-feruloyl)-L-arabinose [x(F)A]. The stability of X(F)A was determined by incubation of (feruloyl-U- 14 C)-labelled X(F)A and (pentosyl-1- 3 H)-labelled X(F)A anaerobically with rat caecal contents and chromatographic analysis of the radioactive products. The ester linkage was hydrolysed very rapidly to form ferulic acid (which was stable) and the disaccharide (XA). The 3 H-XA was further metabolised, but 3 H-monosaccharides did not accumulate. In the end-products of fermentation of (pentosyl- 3 H)-labelled X(F)A, 67% of the 3 H was present in bacterial polymers. In contrast, when free [1- 3 H] arabinose was incubated with rat caecal contents, 74% of the 3 H quickly became volatile, probably as 3 H 2 O. It is concluded that X(F)A is highly susceptible to (feruloyl)esterase activity produced by bacteria present in the rat caecum, and that the disaccharide produced is further fermented, but not via the production of extracellular arabinose and xylose.

Feruloyl and p-coumaroyl groups in spinach cell walls (CW) were labelled using (14C)cinnamic acid and fed to rats. In the caecum and colon, ferulic acid (FA) and p-coumaric acid (PCA) were released from the CW. Few feruloyl or coumaroyl groups remained in the CW to be excreted in faeces, and thus the presence of simple phenol-sugar esters provided little protection of the polysaccharides to enzymic attack. Some oxidatively coupled phenols were also released but a portion remained in the CW. The oxidatively coupled phenols accumulated in the gut whereas the FA and PCA were absorbed by the rat. Thus enzyme-resistant fragments, containing oxidatively coupled phenols (and possibly sugar residues), may survive microbial attack by rat intestinal bacteria.

The aim of this work was to investigate the fate, in the upper gut of a monogastric animal, of polymers bound within plant cell walls. Uniformly and specifically 14 C-labelled spinach cell walls were incubated in artificial body fluids with and without pepsin or pancreatin. In the absence of enzymes, artificial pancreatic juice (pH 8.8) at 37°C hydrolysed the methyl ester groups of wall-bound pectins [half-life (t 1/2 ) 25 h] ; the O-acetyl ester groups of cell wall polysaccharides were much more stable (t 1/2 84 h). In contrast, artificial gastric juice (pH 1.85) hydrolysed wall-bound acetyl groups more rapidly (t 1/2 ∼ 24 h) than methyl ester groups (t 1/2 ∼350 h). Thus, a proportion of the methyl and acetyl groups of plant cell wall polymers will be released in the upper gut as methanol and acetic acid, raising the question of whether these groups should be included within the definition of dietary fibre. The artificial body fluids also caused limited solubilisation of wall polymers but no hydrolysis to mono- or oligosaccharides. Neither pepsin nor pancreatin promoted the hydrolysis of methyl ester or acetyl groups. The small amounts of [ 14 C]protein present in the cell wall preparations were hydrolysed by pepsin or pancreatin to yield amino acids and oligopeptides ; however, the major polysaccharides of spinach cell walls were neither degraded to low-molecular-weight products nor solubilised by these enzymes.

To determine a fate of acetyl groups in intact dietary plant cell walls (PCW) spinach cells were labelled using [1-14C]acetate as a precursor and the PCW isolated. The (acetyl-14C)-PCW were fed to rats by garage and the 14C traced in the rat gut and in the body tissues and excreta of the rat. Some label was solubilised during passage through the upper intestine of the rat, and subsequently incorporated into the body tissues of the rat. A small amount of the radioactivity was excreted as CO2 (4%) and in urine (

The metabolism of dietary plant cell walls (PCW) was followed in the rat using PCW isolated from spinach cell cultures which were14C-labelled in the galacturonic acid residues (using D-[6-14C]glucuronic acid, ⩾85% of the 14C was confined to the C-6 position of pectic galacturonic acid residues) or the methyl ester groups (using L-[methyl-14C]methionine, 90% of the 14C was confined to methyl ester groups and 10% of the 14C in methyl ether groups) of pectin. Eighteen hours after gavage, only 5-10% of the 14C was recovered in the gut contents and faeces. Some of the 14C derived from the [methyl-14C]pectin-labelled PCW was present in the liver (2-4%) and the pelt (6%). In the liver the 14C was mainly in phospholipids, whereas in the pelt the 14C was found in protein (mainly alanine residues) and fatty acids esterified to cholesterol. The livers from rats fed [uronate-6-14C]pectin-labelled PCW contained less 14C (1%) but more 14C was found in the pelt (12-15%). In the liver the 14C was found in phospholipid but in the pelt the label was exclusively found in amino acid residues. Pectin in intact PCW is extensively degraded in the caecum and colon and the products absorbed and metabolised by the host. These products may be important sources of energy and of precursors of structural compounds such as proteins and lipids.

Hemicellulosic polysaccharides from persimmon fruit (Diospyros kaki L.) pericarp were extracted from depectinated cell walls with 0.5, 1 and 4 M KOH at different stages of development: (I) maximal growth corresponding to the first sigmoidal growth phase; (II) cessation of growth corresponding to the lag between the first and the second sigmoidal phases; (III) maximal growth corresponding to the second sigmoidal phase; and (IV) cessation of growth when the fruit had reached its maximum size and the change in colour (green to red) had taken place. During fruit development the amount of total hemicelluloses per unit dry mass cell wall decreased twofold. Xyloglucan was present in the three hemicellulosic fractions, and also decreased with fruit age, although its amount relative to other hemicelluloses increased. The amount of xyloglucan was especially high in the hemicelluloses extracted with 4 M KOH, representing more than 50% at stages III and IV. The average molecular mass of xyloglucan increased from stage I through stage II (0.5 M hemicellulosic fraction) or through stage III (I and 4 M hemicellulosic fractions) and decreased after that. The xyloglucan endotransglycosylase (XET: EC 2.4.1.-) activity was measured as the incorporation of [3H]XXXGol (reduced xyloglucan heptasaccharide labelled at position 1 of the glucitol moiety) into partially purified persimmon fruit xyloglucan. XET specific activity increased greatly between stages I and II. The importance of this enzyme during fruit ripening is discussed.

Using uniformly 14C-labelled spinach (Spinacia oleracea L) plant cell walls (PCW) the metabolism of PCW can be followed in a defined manner in the rat. Only 10% of the 14C was recoverable in the gut contents and faeces 18 h post-gavage. Two percent of the 14C was excreted in urine and 25% excreted as CO2. 14C was found in all tissues of the body but was most concentrated in the adrenal glands, colon and caecum. The 14C was also present at moderately high concentration in the liver pelt, and owing to their larger size, these tissues accounted for a high percentage of the total 14C. In the liver the 14C was predominantly associated with phospholipid, whereas in the pelt it was present in protein and in fatty acid residues esterified to cholesterol. Dietary PCW material is extensively fermented in the caecum and colon. Also, the products of fermentation are a source of energy and important structural precursors of lipids and proteins for the animal.

We have investigated the possibility that extensins (basic, hydroxyproline-rich glycoproteins of the primary cell wall) contribute to cellular adhesion. Soluble [ 14 C]extensin, obtained by salt-elution from the walls of living spinach (Spinacia oleracea L.) cells, was found to adhere strongly to all surfaces tested, including glass, polypropylene and polycarbonate. The adhesion was not prevented by salts or dilute acids, by urea (a neutral chaotropic agent), or by ascorbate (a reducing agent), which indicates that it was not due solely to ionic bonding, hydrogen-bonding or oxidative coupling. The adhesion was prevented by guanidinium thiocyanate, an ionic chaotropic agent, suggesting that ionic bonding and hydrogen-bonding co-operated in the adhesion

A xyloglucan-derived pentasaccharide. Xyl2-Glc3, was shown by viscometry to promote the depolymerisation of xyloglucan by enzyme extracts from bean (Phaseolus vulgaris L. cv. Canadian Wonder) leaves and pea (Pisum sativum L. cv. Alaska) stems. Xyl2-Glc3 was also shown by a radiochemical assay to act as an acceptor substrate for xyloglucan endotransglycosylase activity (XET: EC 2.4.1.—) present in the same extracts. In both these assays, a heptasaccharide (Xyl3-Glc4) was more effective than Xyl2-Glc3 whereas two isomeric tetrasaccharides (Xyl1-Glc3) were essentially ineffective. The agreement in the structural requirements of the two assays suggests that they share a common basis; we therefore propose that the oligosaccharide-sensitive enzyme that depolymerises xyloglucan is XET rather than cellulase (EC 3.2.1.4). In the viscometric assay, the penta- and heptasaccharides would, according to our interpretation, compete with high molecular weight xyloglucan molecules as acceptor substrates for XET, leading to a decrease in the weight-average molecular weight of the xyloglucan and, therefore, to a decrease in viscosity. Our results indicate that oligosaccharides have to possess two α-d-xylose residues in order to act as acceptor substrates for XET. The non-reducing end of a high-molecular weight xyloglucan can also act as an acceptor substrate. Therefore, it is likely that exo-hydrolysis by α-d-xylosidase would destroy the ability of a polysaccharide to act as an acceptor, even though α-d-xylosidase may remove only a single xylose residue from each polysaccharide molecule.

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.

Oxidation of L-[U-14C]tyrosine with 2.7 molar equivalents of alkaline hexacyanoferrate(III) yielded at least 12 chromatographically mobile oxidation products and a large amount of immobile material. Use of 0.23 molar equivalents of hexacyanoferrate(III) yielded [14C]dityrosine (∼1.3% of added [14C]tyrosine) and [14C]isodityrosine (∼0.6%). A chromatographic method is described for the isolation of these two products from the mixture. A method is also described for the prepration of [3H]isodityrosine from non-radioactive isodityrosine by catalytic exchange with 3H2. The [3H]isodityrosine formed was essentially stable in 6M HCI at 110°C, indicating that the tritiation occurred at the benzylic groups.

Oligosaccharides that exert biological effects on higher plants (other than as carbon or energy sources) have been termed‘oligosaccharins'. A limited number of specific oligo-β-glucans derived from fungal cell walls exhibit oligosaccharin activity, at very low doses, switching on the synthesis of phytoalexins. The structural requirements for this biological activity are stringent, and there is strong evidence that the oligosaccharins of fungal origin act through a receptor in the plant cell membrane. Chemically unrelated oligosaccharins can also be produced by partial digestion of pectins and xyloglucans from higher plant cell walls. In some cases, for example, the α-l-fucosylated oligoxyloglucans that antagonise the growth-promoting effect of auxin, these plant-derived oligosaccharins have relatively strict structural requirements for activity and act at ∼10−6 mol m−3; these may act via specific receptors. In other cases, for example, the growth-promoting action of other oligoxyloglucans, the stringency is somewhat lower and the activity is only seen at ∼10−3 mol m−3, and these oligosaccharins are proposed to influence growth through their ability to modulate the activity of an enzyme, cellulase. A third class of plant-derived oligosaccharins, the oligo-α-galacturonides, appear to have much less stringent structural requirements; a number of unrelated physiological responses are evoked by the same range of oligo-α-galacturonides at ∼10−3 to 1 mol m−3. We suggest that these oligosaccharins may act via a mechanism not involving a specific receptor, perhaps by interacting with the plasma membrane to bring about a change in its physical properties.

A protocol is described which allows the detection of transglycosylase isoenzyme activity. Crude extracts of cauliflower (Brassica oleracea var. botrytis) florets were treated with 1 M sodium chloride, filtered through cellulose acetate, dialysed, concentrated and subjected to isoelectric focusing under non-denaturing conditions in a polyacrylamide gel. After electrophoresis, the gel was overlaid with a test paper impregnated with a mixture of high-Mr xyloglucan and a sulphorhodamine-conjugated oligosaccharide of xyloglucan. Transglycosylation caused the production of sulphorhodamine-labelled polysaccharide, which remained bound to the test paper after prolonged washing in water, revealing eight fluorescent bands corresponding to XET isoenzymes with pI values of 8.8, 8.6, 7.8, 7.6, 7.3, 7.1, 6.7 and 6.6. Copyright © 1999 John Wiley & Sons, Ltd.

Xyloglucan-oligosaccharides and cello-oligosaccharides, both of which are potential products of the action of cellulase on plant cell wail polysaccharides, inhibited acid-induced elongation in pea (Pisum sativum L. cv. Alaska) stem segments. Xyloglucan-derived nonasaccharide (XG9; Glc4-Xyl3)Gal-Fuc) and decasaccharide (XG10; Glc4-Xyl3-Gal2-Fue) inhibited acid-induced growth at 1.0 and 0.1 nM, respectively, whereas the heptasaccharide (XG7; Glc4-Xyl3) and octasaccharide (XG8; Glc4-Xyl3-Gal)2 which lack L-fucose, did not. XG9 at 1 nM inhibited acid-induced growth as effectively as it inhibits auxin-induced elongation. This suggests that XG9's effect as an inhibitor of auxin action is not mediated by a suppresion of H+-efflux, but rather that XG9 blocks some step that is common to the action of both auxin and H+ on growth. Cello-oligosaccharides (degree of polymerisation 4–7) also inhibited acid-induced growth at 10 nM; these are therefore a new class of possible oligosaccha-rin. The inhibitory effect of xyloglucan- and cellooligosaccharides on acid-induced growth was rapidly reversed by washing.

A revised system of abbreviated names is proposed for xyloglucan-derived oligosaccharides. Each (1→4)-linked β-D-glucosyl residue (and the reducing terminal n-glucose moiety) of the backbone is given a one-letter code according to its substituents. The name of the oligosaccharide consists of these code letters listed in sequence from non-reducing to reducing terminus of the backbone

The cell wall, as the outer coat of a plant cell, controls cell expansion, gives plant cells their characteristic shapes, and provides protection. Cellulose is only one of many polysaccharides and other polymers of the higher plant cell wall; some of these polymers undergo chemical reactions in the walls of living cells. Keywords: cell wall; polysaccharide; growth (plant); epidermis (plant); crosslinks

Most plant cell wall polymers are synthesized from ‘activated’ precursors by the action of transferases. These enzymes are located in cell membranes (synthesizing polysaccharides and glycoproteins) or in the cell wall itself (synthesizing cutin and suberin). Lignin, in contrast, is synthesized in the wall by oxidative rather than transferase-catalysed reactions. Keywords: cell wall polymer assembly; polysaccharides (plant); Golgi bodies; nucleoside diphosphate sugars; lignin

SUMMARY Hypocotyls of embryos from unripe and soaked ripe seeds of Phaseolus vulgaris and Acer pseudoplatanus are shown to be capable of transporting auxin. The transport is strongly polar and shows the characteristics of active transport in the basipetal but not the acropetal direction. A procambial cylinder and a few phloem elements have been formed in these hypocotyls.

The plant growth promoter, auxin, may loosen the primary cell wall by increasing the activity of extracellular cellulases – a group of enzymes that cleave hemicellulose chains in the walls of both monocotyledons and dicotyledons. Evidence is reviewed that suggests that these hemicellulose chains tether adjacent microfibrils, and that by cleaving such chains the cellulases facilitate cell expansion. On the basis of this structural arrangement a mechanism for elastic and plastic wall extension is proposed.