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We established model systems for exploring the roles of symplastic and apoplastic ascorbate in heavy-metal-polluted dicot and monocot cells. Cell-suspension cultures of Arabidopsis and maize were treated with copper, cadmium or nickel; growth and ascorbate metabolism were measured. Growth was halved by ∼80 µM Cu2+, 90 µM Cd2+ or 1200 µM Ni2+ in Arabidopsis, and ∼90 µM Cu2+, 650 µM Cd2+ or 650 µM Ni2+ in maize. Cu2+ (128 µM) and Cd2+ (512 µM) caused partial loss of symplastic ascorbate, especially in Arabidopsis; Ni2+ (512 and 2048 µM) had moderate effects. Added apoplastic l-ascorbate (1 mM) was consumed by the cultures (half-life ∼23 and 44 min in Arabidopsis and maize, respectively), consumption rate being 3–6-fold increased by Cu2+, Cd2+ and Ni2+ in Arabidopsis, and by Cu2+ in maize; Cd2+ and Ni2+ had relatively little effect on apoplastic ascorbate consumption in maize. Radioactivity from exogenous 1 mM l-[1-14C]ascorbate remained extracellular; catabolites formed were dehydroascorbic acid, diketogulonate and oxalyl-threonates. In conclusion, suspension-cultured cells respond to heavy-metal stresses by maintaining symplastic ascorbate concentrations, which may beneficially scavenge symplastic reactive oxygen species (ROS). Apoplastic ascorbate is catabolised in metal-polluted cultures via several oxidative and non-oxidative reactions, the former potentially scavenging stress-related apoplastic ROS. [ABSTRACT FROM AUTHOR]

Summary: Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE‐TO‐FREEZING8, in Arabidopsis thaliana.We identified SFR8 using recombination‐based mapping and whole‐genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC‐MS and polyacrylamide gel electrophoresis to measure cell‐wall fucose and boron (B)‐dependent dimerization of the cell‐wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate‐bridging of RGII, we assessed freeze‐induced damage in wild‐type and sfr8 plants by measuring electrolyte leakage from freeze‐thawed leaf discs.We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP‐d‐mannose‐4,6‐dehydratase. sfr8 cell walls exhibited low cell‐wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing‐sensitive.Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize. [ABSTRACT FROM AUTHOR]

Background and Aims Xyloglucan endotransglucosylase/hydrolase (XTH) proteins that possess xyloglucan endotransglucosylase (XET) activity contribute to cell-wall assembly and remodelling, orchestrating plant growth and development. Little is known about in-vivo XET regulation, other than at the XTH transcriptional level. Plants contain 'cold-water-extractable, heat-stable polymers' (CHPs) which are XTH-activating factors (XAFs) that desorb and thereby activate wall-bound XTHs. Because XAFs may control cell-wall modification in vivo , we have further explored their nature. Methods Material was cold-water-extracted from 25 plant species; proteins were precipitated by heat-denaturation, then CHP was ethanol-precipitated. For XAF assays, CHP (or sub-fractions thereof) was applied to washed Arabidopsis thaliana cell walls, and the enzymes thus solubilized were assayed radiochemically for XET activity. In some experiments, the CHP was pre-treated with trifluoroacetic acid (TFA), alkali (NaOH) or glycanases. Key Results CHP specifically desorbed wall-bound XTHs, but not β-glucosidases, phosphatases or peroxidases. CHP preparations from 25 angiosperms all possessed XAF activity but had no consistent monosaccharide composition. Of 11 individual plant polymers tested, only gum arabic and tamarind xyloglucan were XAF-active, albeit less so than CHP. On gel-permeation chromatography, XAF-active cauliflower CHP eluted with a molecular weight of ~7000–140 000, although no specific sugar residue(s) co-eluted exactly with XAF activity. Cauliflower XAF activity survived cold alkali and warm dilute TFA (which break ester and glycofuranosyl linkages, respectively), but was inactivated by hot 2 m TFA (which breaks glycopyranosyl linkages). Cauliflower XAF activity was remarkably stable to diverse glycanases and glycosidases. Conclusions XAFs are naturally occurring heat-stable polymers that specifically desorb (thereby activating) wall-bound XTHs. Their XAF activity considerably exceeds that of gum arabic and tamarind xyloglucan, and they were not identifiable as any major plant polysaccharide. We propose that XAF is a specific, minor, plant polymer that regulates xyloglucan transglycosylation in vivo , and thus wall assembly and restructuring. [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]

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage β-glucan. Isoforms differed in optimum pH (5.0–7.5), in temperature dependence and in acceptor substrate preferences. [ABSTRACT FROM PUBLISHER]

•Apoplastic ascorbate has been proposed to confer resistance to oxidative stresses, e.g. ozone. We investigated reactive oxygen species (ROS)-induced secretion and catabolism of ascorbate. •Late-growth-phase cultured cells of rose and Arabidopsis were preloaded with [14C]ascorbate. Radiolabelled metabolites and secretion products were analysed by high-voltage electrophoresis. •In both species, exogenous 1 mM hydrogen peroxide (H2O2) rapidly stimulated [14C]ascorbate and [14C]dehydroascorbate accumulation in the medium (apoplast). Net 14C export was most rapid within 100 s of washing, and often showed superimposed pulses, of c. 10-s duration, whose amplitude was greater after H2O2 treatment. Oxidative stress did not cause indiscriminate metabolite leakage from the cells. H2O2 caused c. 20–40% of the intracellular [14C]ascorbate to be irreversibly catabolized to [14C]oxalyl-threonate and related products; however, the great majority of the extracellular radioactivity remained as [14C]ascorbate and [14C]dehydroascorbate. Much of the apoplastic dehydroascorbate was probably reabsorbed by the cells and reduced back to ascorbate. •The data show that exported ascorbate can serve an apoplastic antioxidant role in these late-growth-phase cells without being irreversibly lost, whereas in early-growth-phase cells most extracellular ascorbate is irreversibly degraded. In conclusion, cultured plant cells can respond actively to oxidative stress by reversibly exporting ascorbate into the apoplast. [ABSTRACT FROM AUTHOR]

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans β-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition. [ABSTRACT FROM PUBLISHER]

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture. [Copyright &y& Elsevier]

Xyloglucan plays an important structural role in primary cell walls, possibly tethering adjacent microfibrils and restraining cell expansion. There is therefore considerable interest in understanding the role of xyloglucan endotransglucosylase/hydrolases (XTHs), which are encoded in Arabidopsis by a 33-member gene family. We compared the key catalytic properties of two very different Arabidopsis XTHs (heterologously produced in Pichia ), both of which are aluminium-repressed. Reductively tritiated oligosaccharides of xyloglucan were used as model acceptor substrates. Untransformed Pichia produced no xyloglucan-acting enzymes; therefore purification of the XTHs was unnecessary. XTH15, a classical group-I/II XTH, had high XET and undetectable XEH activity in vitro ; its XET K m values were 31 μM XXXGol (acceptor substrate) and 2.9 mg/ml xyloglucan (donor substrate). In contrast, XTH31, a group-III-A XTH, showed predominant XEH activity and only slight XET activity in vitro ; its XET K m was 86 μM XXXGol (acceptor), indicating a low affinity of this predominantly hydrolytic protein for a transglycosylation acceptor substrate. The K m of XTH31’s XEH activity was 1.6 mg/ml xyloglucan. For both proteins, the preferred XET acceptor substrate, among five cellotetraitol-based oligosaccharides tested, was XXXGol. XTH31’s XET activity was strongly compromised when the second Xyl residue was galactosylated. XTH15’s XET activity, in contrast, tolerated substitution at the second Xyl residue. The two enzymes also showed different pH preferences, XTH31 exhibiting an unusually low pH optimum and XTH15 an unusually broad optimum. XTH31’s hydrolase activity increased almost linearly with decreasing pH in the apoplastic range, 6.2–4.5, consistent with a possible role in ‘acid growth’. In conclusion, these two Al 3+ -repressed XTHs differ, in several important enzymic features, from other members of the Arabidopsis XTH family. [ABSTRACT FROM AUTHOR]

Abstract: Two new reducing glycoconjugates [N-d-galacturonoyl-putrescinamide (GalA–Put) and N,N′-di-d-galacturonoyl-putrescinamide (GalA–Put–GalA)] and homogalacturonan–putrescine (GalA n –Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25°C) but rapidly hydrolysed by these agents at 100°C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalA n –Put, releasing fragments such as GalA3–Put–GalA3. To trace the possible formation of GalA–polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional ∼73% of the 14C taken up (in Arabidopsis), or ∼21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide–Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin–polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes. [Copyright &y& Elsevier]