Xyloglucan Endotransglucosylase Activity Research Articles

Genome-wide analysis of the XTH gene family and functional analysis of DlXTH23.5/25 during early longan somatic embryogenesis.

Xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is a cell wall-modifying protein that affects cell expansion and loosening of the cell wall. This study focused on the regulatory mechanism of DlXTH genes during early somatic embryogenesis (SE) and the heat stress response in longan. Mining of the available D. longan genome sequence yielded 25 putative XTH genes. Transcript profiles based on RNA sequencing (RNA-seq) data showed that most of the 17 detected DlXTH genes were highly expressed in the embryogenic callus (EC) (8) and globular embryo (GE) (8), and 13 of them responded significantly to heat stress. The assay for transposase-accessible chromatin sequencing (ATAC-seq) data analysis showed that in terms of chromatin accessibility, 22 of the 25 DlXTH genes were open during early SE, and most of the peak DlXTH genes with transcription differences during early SE were associated with high levels of H3K4me1. The most differentially expressed genes, DlXTH23.5 and DlXTH25, were selected for analysis. According to subcellular localization and quantitative real-time PCR (qRT-PCR) analysis, DlXTH23.5/25, which encode cell membrane-localized proteins, were expressed at the highest level in the GE and significantly responded to heat stress. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY31, DlERF1, and DlERF5 might bind to the DlXTH23.5/25 promoters to activate gene transcription. Transient overexpression of TFs and DlXTH23.5/25 induced XET activity in Nicotiana benthamiana leaves. Under heat stress in the longan EC, the XET activities and expression levels of TFs and DlXTH23.5/25 were significantly increased, and a high concentration of XET might inhibit longan SE. Thus, the regulatory network composed of DlXTH23.5/25 and its related TFs may regulate early longan SE and participate in the regulatory pathway of longan under heat stress via cell wall repair through the action of XET.

Identification and functional characterization of the xyloglucan endotransglucosylase/hydrolase 32 (AhXTH32) in peanut during aluminum-induced programmed cell death

The toxicity of aluminum (Al) in acidic soil is a prevalent problem and causes reduced crop yields. In the plant response to Al toxicity, programmed cell death (PCD) appears to be an important mechanism. The plant cell wall of crop roots is the predominant site targeted by Al. Here, studies of the capacities of different cell wall constituents (pectin, hemicellulose 1 {HC1} and HC2) to adsorb Al indicated that HC1 has the greater ability to bind Al. The activity of xyloglucan endotransglucosylase (XET) was significantly inhibited by Al in the Al-tolerant peanut cultivar ‘99–1507’ compared to that in ‘ZH 2’ (Al-sensitive). Results from qPCR analysis suggested that the suppression of XET activity by Al was transcriptionally regulated and that xyloglucan endotransglucosylase/hydrolase 32 (AhXTH32) was the major contributor to these changes. The overexpression of AhXTH32 in Arabidopsis strongly inhibited root growth with a loss of viability in root cells and the occurrence of typical hallmarks of PCD, while largely opposite effects were observed after xth32 suppression. AhXTH32 contributed to the modulation XET and xyloglucan endohydrolase (XEH) activity in vivo. Taken together, our results demonstrate that Al-tolerant peanut cultivar root tips cell walls bind Al predominantly in the HC1 fraction, which results in the inhibition of AhXTH32, with consequences to root growth, Al sensitivity, the occurrence of PCD and the XET/XEH activity ratio.

Isolation and characterization of Populus xyloglucan endotransglycosylase/hydrolase (XTH) involved in osmotic stress responses

Xyloglucan endotransglycosylase/hydrolase (XTH) belongs to the GH16 subfamily of the glycoside hydrolases of carbohydrate active enzymes and plays an important role in the structure and function of plant cell walls. In this study, 11 members of the XTH gene family were cloned from Populus tomentosa. A bioinformatics analysis revealed that 11 PtoXTHs could be classified into three groups, where PtoXTH27 and PtoXTH34 were most likely to exhibit XTH activity. Biochemical analyses of purified PtoXTHs demonstrated that PtoXTH27 and PtoXTH34 had detectable xyloglucan endotransglucosylase (XET) activity, while the others did not exhibit XET or XEH activity. Moreover, enzymatic assays revealed that the optimum reaction temperature of both PtoXTH27 and PtoXTH34 was 37 °C, while their optimum pH values differed, such that PtoXTH27 was 6.0 and PtoXTH34 was 5.0. Enzyme kinetic parameters indicated that PtoXTH34 had higher affinity for the receptor substrate, XXXG, implying that PtoXTH34 and PtoXTH27 in plants have different substrate structure specificity. Finally, heterologous expression of XTH significantly increased intracellular total sugar content and osmotolerance of yeast cells, indicating that PtoXTH27 and PtoXTH34 are potentially involved in osmotic stress responses. These results clearly demonstrate the enzymatic characteristics and putative role of XTH in osmotic stress responses.

Functional and chemical characterization of XAF: a heat-stable plant polymer that activates xyloglucan endotransglucosylase/hydrolase (XTH).

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. 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. 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. 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.

FeSTAR2 interacted by FeSTAR1 alters its subcellular location and regulates Al tolerance in buckwheat

Buckwheat (Fagopyrum esculentum) exhibits high Al tolerance, but only a few genes have been functionally characterized. We previously characterized a half-type ABC transporter, Fagopyrum esculentum sensitive to Al rhizotoxicity1 (FeSTAR1), in buckwheat Al tolerance. This study aims to investigate whether and how another half-type ABC transporter (FeSTAR2) plays role in Al tolerance in buckwheat. The expression of FeSTAR2 and complementation test in Arabidopsis als3 mutant was examined. The interaction between FeSTAR1 and FeSTAR2 and subcellular location were analyzed by bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays. Al rapidly and specifically induced FeSTAR2 expression. Having transmembrane domains, FeSTAR2 localizes to membrane. BiFC and Y2H assays showed that FeSTAR2 could interact with FeSTAR1 which contains only nucleotide binding domain. Intriguingly, interaction between FeSTAR1 and FeSTAR2 altered their locations. Both heterologous expression of FeSTAR2 in als3 and exogenous UDP-glucose rescued its Al hypersensitivity of als3 mutant, suggesting that involvement of FeSTAR2 in Al tolerance requires UDP-glucose. Furthermore, Al-induced inhibition of xyloglucan endotransglucosylase (XET) activity in both atstar1 and als3 mutants could be restored by UDP-glucose. Our results indicate that FeSTAR2 interacts with FeSTAR1 to form an ABC transporter to regulate Al tolerance by vesicular transport of UDP-glucose which affects hemicellulose metabolism by regulating XET activity.

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation.

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium distachyon transcriptional profiling data, we identified a B distachyon trihelix family transcription factor (BdTHX1) that is highly coexpressed with the B distachyon CSLF6 gene (BdCSLF6), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature B distachyon seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of BdCSLF6 as one of the most enriched sequences. The binding of BdTHX1 to the BdCSLF6 intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase (BdXTH8) is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. B distachyon shoots regenerated from transformed calli overexpressing BdTHX1 showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8.

Nitric Oxide Inhibits Al-Induced Programmed Cell Death in Root Tips of Peanut (Arachis hypogaea L.) by Affecting Physiological Properties of Antioxidants Systems and Cell Wall.

It has been reported that nitric oxide (NO) is a negative regulator of aluminum (Al)-induced programmed cell death (PCD) in peanut root tips. However, the inhibiting mechanism of NO on Al-induced PCD is unclear. In order to investigate the mechanism by which NO inhibits Al-induced PCD, the effects of co-treatment Al with the exogenous NO donor or the NO-specific scavenger on peanut root tips, the physiological properties of antioxidants systems and cell wall (CW) in root tip cells of NO inhibiting Al-induced PCD were studied with two peanut cultivars. The results showed that Al exposure induced endogenous NO accumulation, and endogenous NO burst increased antioxidant enzyme activity in response to Al stress. The addition of NO donor sodium nitroprusside (SNP) relieved Al-induced root elongation inhibition, cell death and Al adsorption in CW, as well as oxidative damage and ROS accumulation. Furthermore, co-treatment with the exogenous NO donor decreased MDA content, LOX activity and pectin methylesterase (PME) activity, increased xyloglucan endotransglucosylase (XET) activity and relative expression of the xyloglucan endotransglucosylase/hydrolase (XTH-32) gene. Taken together, exogenous NO alleviated Al-induced PCD by inhibiting Al adsorption in CW, enhancing antioxidant defense and reducing peroxidation of membrane lipids, alleviating the inhibition of Al on root elongation by maintaining the extensibility of CW, decreasing PME activity, and increasing XET activity and relative XTH-32 expression of CW.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets

Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo.

Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29μM), retinoic acid, eosin (IC50 27μM) and erythrosin (IC50 36μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads.

Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis.

Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.

XTH31, encoding an in vitro XEH/XET-active enzyme, regulates aluminum sensitivity by modulating in vivo XET action, cell wall xyloglucan content, and aluminum binding capacity in Arabidopsis.

Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of (27)Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity.

Mixed‐linkage glucan:xyloglucan endotransglucosylase (MXE) re‐models hemicelluloses in Equisetum shoots but not in barley shoots or Equisetum callus

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 [(3)H]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.

English

Biomass production in plant is directly related to the amount of intercepted solar radiation by the canopy and available water to the plant. Growth and development of leaves, especially under drought condition, is therefore major determinant of crop productivity. Xyloglucan endotransglucosylase (XET) plays important role in growth and development of plants. XETs are a family of enzymes that mediate construction and restructuring of xyloglucan cross-links, thereby controlling the mechanical properties of cell wall. We cloned complete cDNA of an XET from pearl millet (Pennisetum glaucum L.) and characterized it using in silico comparative genomics and activity assays. The cloned cDNA was 1266 bp in length, encoding a protein with 291 amino acids having signal peptide targeting it to the cell wall. The protein showed xyloglucan endotransglucosylase activity but no hydrolytic activity, therefore, named as PgXET1 as per the convention. The comparative genomics revealed that the functional sites of the enzyme (XET) were highly conserved. Evolutionary studies using phylogenetic tree indicated its grouping with XETs from maize (with >95% bootstrap support), barley, rice, etc. This is the first report on cloning and characterization of an XET (PgXET1) from pearl millet, an important dual-purpose crop.   Key words:    Xyloglucan endotransglucosylase, Pennisetum glaucum, pearl millet, primary cell wall, cell expansion, drought tolerance.

Cell wall hemicellulose contributes significantly to aluminum adsorption and root growth in Arabidopsis.

The cell wall (CW) has been recognized as the major target of aluminum (Al) toxicity. However, the components responsible for Al accumulation and the mechanisms of Al-induced CW function disruption are still elusive. The contribution of different CW components (pectin, hemicellulose 1 [HC1], and HC2) to adsorb Al and the effect of Al on xyloglucan endotransglucosylase/hydrolyase activity were investigated in Arabidopsis (Arabidopsis thaliana) in this study. A fractionation procedure was optimized to effectively extract different CW components, especially to prevent the HC fraction from pectin contamination. When CW materials extracted from Al-treated roots (50 μm Al for 24 h) were fractionated, about 75% of CW Al accumulated in the HC1 fraction. A time-dependent kinetic study showed that only when the HC1 fraction was removed was the amount of Al adsorbed decreased sharply. In vivo localization of xyloglucan endotransglucosylase (XET) activity showed that Al greatly inhibited this enzyme activity within 30 min of exposure, which was concomitant with Al-induced callose deposition in roots. Results from real-time reverse transcription-polymerase chain reaction indicated that three genes may constitute the major contributors to XET activity and that the inhibition of XET activity by Al is caused by transcriptional regulation. These results, to our knowledge for the first time, demonstrate that HC is the major pool for Al accumulation. Furthermore, Al-induced reduction in XET activity could play an important role in Al-induced root growth inhibition.

Biological implications of the occurrence of 32 members of the XTH (xyloglucan endotransglucosylase/hydrolase) family of proteins in the bryophyte Physcomitrella patens

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi-gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P.patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P.patens, constituting a fairly large multi-gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P.patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue-dependent. However, comparison of structural features of families of XTH genes between P.patens and angiosperms demonstrated the existence of several bryophyte-specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte-specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non-seed plants.

Xyloglucan: the molecular muscle of trees.

Tension wood evolved in woody angiosperms to allow stems with secondary thickening to bend and thus maintain an optimal orientation. Stem bending is the result of longitudinal tensile stress that develops in tension wood tissues. In many species, a specialized secondary cell wall layer, the so-called gelatinous (G)-layer, develops, containing longitudinally orientated crystalline cellulose fibrils; these have been recently shown to generate the tensile stress by an unknown mechanism. The cellulose fibrils cannot, however, work in isolation. Both coherence between the fibrils and adherence of the G-layer to the adjacent cell wall layers are required to transfer the tensile stresses of the cellulose fibrils to the tissue. Previous work had not identified hemicelluloses within the G-layer. Sugar composition and polysaccharide linkage analyses of pure G-layers isolated by sonication have recently identified xyloglucan as the main non-cellulosic component of the G-layer. Xyloglucan has been detected by immunolabelling with the CCRC-M1 monoclonal antibody and by in-situ activity assays using XXXG-sulforhodamine substrate in the developing G-layers but not in the mature ones. However, xyloglucan endotransglucosylase/hydrolase (XTH) proteins persist in the G-layer for several years and the corresponding xyloglucan endotransglucosylase (XET) activity (EC 2.4.1.207) occurs in the adjacent layers. Correspondingly, several XTH-encoding transcripts were found to be up-regulated in developing tension wood compared with normal wood. We propose that, during cellulose crystallization, a part of the xyloglucan is trapped inside the crystal, inducing longitudinal tensile stress within it; another part of it is accessible and present between the G-layer and the outer wall layers. XET activity that occurs persistently in the G-fibres maintains coherence between the G-layer and the adjacent secondary wall layers. It is postulated that these activities are essential for generation of tensile stress during fibre maturation in tension wood.

Analysis of xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and apple

Xyloglucan endotransglucosylase/hydrolase (XTH) enzymes are thought to play a key role in fruit ripening by loosening the cell wall in preparation for further modification by other cell wall-associated enzymes and through disassembly of xyloglucan. Twenty-five XTH genes were isolated and characterised from kiwifruit and apple databases containing >270,000 expressed sequence tags (ESTs). The XTH genes (14 from kiwifruit, 11 from apple) encoded putative proteins with similar molecular weights, but with isoelectric points ranging from acidic to basic. All 25 XTH amino acid sequences contained the two conserved glutamic acid residues at the active site of the XTH enzyme. Phylogenetic analysis produced trees with branches each containing kiwifruit, apple, tomato and Arabidopsis XTH sequences, suggesting the potential for functional orthology. EST expression profiling and quantitative PCR analysis were used to identify kiwifruit and apple XTH transcripts expressed in root tip, flower, young leaf and ripe fruit. In ripe apple fruit, the transcripts for two XTH genes ( Md-XTH2 and Md-XTH10) were the most abundant, whilst in ripe kiwifruit three transcripts predominated ( Ad-XTH4, 5 and 7). Ad-XTH7 was highly expressed in the outer pericarp of firm kiwifruit at commercial harvest and expression of this mRNA decreased during the rapid softening phase. Three kiwifruit XTH genes ( Ad-XTH5, Ad-XTH7 and Ac-XTH14) were expressed in Escherichia coli and the recombinant proteins were shown to have xyloglucan endotransglucosylase activity. This research lays the groundwork for understanding the role of XTHs during fruit ripening and storage in kiwifruit and apple.

Mixed‐linkage β‐glucan : xyloglucan endotransglucosylase, a novel wall‐remodelling enzyme from Equisetum (horsetails) and charophytic algae

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.

Overexpression of Poplar Cellulase Accelerates Growth and Disturbs the Closing Movements of Leaves in Sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

Anionic derivatives of xyloglucan function as acceptor but not donor substrates for xyloglucan endotransglucosylase activity

Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic acids, deduced to be beta-D: -GalpA-(1-->2)-D-Xyl and beta-D: -GlcpA-(1-->4)-D-Glc. Anionic xyloglucan had a decreased ability to hydrogen-bond to cellulose and to complex with iodine. It was almost totally resistant to digestion by cellulase [endo-(1-->4)-beta-glucanase] and did not serve as a donor substrate for xyloglucan endotransglucosylase (XET) activity. Like several other anionic polysaccharides, it promoted XET activity when unmodified (non-ionic) xyloglucan was used as donor substrate. Anionic xyloglucan may mimic polyanions whose presence in the plant cell wall promotes the action of endogenous XTH proteins. NaOCl with TEMPO oxidised the heptasaccharide, XXXG, to form XXX-glucarate, which did serve as an acceptor substrate although at a rate approximately fourfold less than XXXG itself. Anionic derivatives of xyloglucan, acting as acceptor but not donor substrates, may be valuable tools for exploring the biological roles of XTHs in the integration versus the re-structuring of xyloglucan in the plant cell wall.