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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.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Background Xyloglucan endotransglycosylase/hydrolases (XTH) can disrupt and reconnect the xyloglucan chains, modify the cellulose-xyloglucan complex structure in the cell wall to reconstruct the cell wall. Previous studies have reported that XTH plays a key role in the aluminum (Al) tolerance of tea plants (Camellia sinensis), which is a typical plant that accumulates Al and fluoride (F), but its role in F resistance has not been reported. Results Here, 14 CsXTH genes were identified from C. sinensis and named as CsXTH1–14. The phylogenetic analysis revealed that CsXTH members were divided into 3 subclasses, and conserved motif analysis showed that all these members included catalytic active region. Furthermore, the expressions of all CsXTH genes showed tissue-specific and were regulated by Al3+ and F− treatments. CsXTH1, CsXTH4, CsXTH6–8 and CsXTH11–14 were up-regulated under Al3+ treatments; CsXTH1–10 and CsXTH12–14 responded to different concentrations of F− treatments. The content of xyloglucan oligosaccharide determined by immunofluorescence labeling increased to the highest level at low concentrations of Al3+ or F− treatments (0.4 mM Al3+ or 8 mg/L F−), accompanying by the activity of XET (Xyloglucan endotransglucosylase) peaked. Conclusion In conclusion, CsXTH activities were regulated by Al or F via controlling the expressions of CsXTH genes and the content of xyloglucan oligosaccharide in C. sinensis roots was affected by Al or F, which might finally influence the elongation of roots and the growth of plants.

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

Plants have evolved various strategies to sense and respond to saline environments, which severely reduce plant growth and limit agricultural productivity. Alteration to the cell wall is one strategy that helps plants adapt to salt stress. However, the physiological mechanism of how the cell wall components respond to salt stress is not fully understood. Here, we show that expression of XTH30, encoding xyloglucan endotransglucosylase-hydrolase30, is strongly up-regulated in response to salt stress in Arabidopsis. Loss-of-function of XTH30 leads to increased salt tolerance and overexpression of XTH30 results in salt hypersensitivity. XTH30 is located in the plasma membrane and is highly expressed in the root, flower, stem, and etiolated hypocotyl. The NaCl-induced increase in xyloglucan (XyG)-derived oligosaccharide (XLFG) of the wild type is partly blocked in xth30 mutants. Loss-of-function of XTH30 slows down the decrease of crystalline cellulose content and the depolymerization of microtubules caused by salt stress. Moreover, lower Na+ accumulation in shoot and lower H2O2 content are found in xth30 mutants in response to salt stress. Taken together, these results indicate that XTH30 modulates XyG side chains, altered abundance of XLFG, cellulose synthesis, and cortical microtubule stability, and negatively affecting salt tolerance., Xyloglucan endotransglucosylase-hydrolase30 modulates xyloglucan side chains by altering abundance of xyloglucan-derived oligosaccharide, negatively affecting salt tolerance.

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-β-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins’ enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXEand CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.

The notion that xyloglucans (XG) play a pivotal role in tethering cellulose microfibrils in the primary cell wall of plants can be traced back to the first molecular model of the cell wall proposed in 1973, which was reinforced in the 1990s by the identification of Xyloglucan Endotransglucosylase/Hydrolase (XTH) enzymes that cleave and reconnect xyloglucan crosslinks in the cell wall. However, this tethered network model has been seriously challenged since 2008 by the identification of the Arabidopsis thaliana xyloglucan-deficient mutant (xxt1 xxt2), which exhibits functional cell walls. Thus, the molecular mechanism underlying the physical integration of cellulose microfibrils into the cell wall remains controversial. To resolve this dilemma, we investigated the cell wall regeneration process using mesophyll protoplasts derived from xxt1 xxt2 mutant leaves. Imaging analysis revealed only a slight difference in the structure of cellulose microfibril network between xxt1 xxt2 and wild-type (WT) protoplasts. Additionally, exogenous xyloglucan application did not alter the cellulose deposition patterns or mechanical stability of xxt1 xxt2 mutant protoplasts. These results indicate that xyloglucan is not essential for the initial assembly of the cellulose network, and the cellulose network formed in the absence of xyloglucan provides sufficient tensile strength to the primary cell wall regenerated from protoplasts.

The role of xyloglucan endotransglycosylases in the regulation and promotion of plant growth in response to such widespread stress factors as drought, salinization, and hypothermia remains poorly understood. The tXET-B2 (SlXTH10) gene encodes one of the xyloglucan endotransglycosylases of tomato, which is most closely related in the nucleotide sequence to the AtXTH15 and AtXTH16 genes of Arabidopsis thaliana. At present, the specific functions of the tXET-B2 gene, as well as of its homologs, AtXTH15 and AtXTH16, remain obscure. To study the role of tXET-B2 in the regulation of growth and adaptation to abiotic stress factors, transgenic tobacco plants with estradiol-inducible expression of the tomato tXET-B2 gene were generated. Overexpression of this gene promoted tobacco root growth in a medium containing 50 mM NaCl. Under drought conditions, exogenous treatment with estradiol resulted in a considerable increase in fresh and dry weight in many of the studied transgenic lines. Under normal conditions, as well as under salinization and hypothermia stress, such positive effect was detected only for some transgenic lines. The obtained data point to the possibility of using genetically engineered constructs of the tXET-B2 gene to correct growth parameters of transgenic plants under the influence of stress factors.

Aluminum (Al) toxicity is one of the primary factors limiting crop production in acid soils worldwide. The cell wall is the major target of Al toxicity owing to the presence of many Al binding sites. Previous studies have found that XTH, encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), could participate in cell wall extension and affect the binding ability of the cell wall to Al by impeding the activities of these two enzymes. In this study, we found that ZmXTH, an XTH gene in maize, was involved in Al detoxification. The Al-induced up-regulation of ZmXTH occurred in the roots, prominently in the root tips. Additionally, the expression of ZmXTH was specifically induced by Al3+ but no other divalent or trivalent cations. Compared with the wild-type Arabidopsis, ZmXTH overexpressing plants grew more healthy and had decreased Al content in their root and root cell wall after Al stress. Overall, the results suggest that ZmXTH could confer the Al tolerance of transgenic Arabidopsis plants by reducing the Al accumulation in their roots and cell walls.

Background The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. Results A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. Conclusions A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits. Electronic supplementary material The online version of this article (10.1186/s13068-017-0991-x) contains supplementary material, which is available to authorized users.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage β-d-glucan (MLG). However, Equisetum hetero-trans-β-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) – and, we now show, xyloglucan polysaccharide – in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose–xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance., Studying a hetero-transglucanase showed that cellulose and mixed-linkage glucan can be covalently linked to the hemicellulose xyloglucan in native plant cell walls. These reactions increase with tissue age and predominate in structural tissues and in cells surrounding cavities, suggesting a role in tissue strengthening. Expansin strongly augments cellulose hetero-transglucosylation but has no effect on mixed-linkage glucan.

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

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. Since 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 enzymes thus solubilised 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 eleven 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 molecular weight ~7,000–140,000, though 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 2M 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

Xyloglucan endotransglucosylase/hydrolases (XTHs)&mdash, a family of xyloglucan modifying enzymes&mdash, play an essential role in the construction and restructuring of xyloglucan cross-links. However, no comprehensive study has been performed on this gene family in barley. A total of 24 HvXTH genes (named HvXTH1-24) and an EG16 member were identified using the recently completed genomic database of barley (Hordeum vulgare). Phylogenetic analysis showed that 24 HvXTH genes could be classified into three phylogenetic groups: (I/II, III-A and III-B) and HvXTH15 was in the ancestral group. All HvXTH protein members&mdash, except HvXTH15&mdash, had a conserved N-glycosylation site. The genomic location of HvXTHs on barley chromosomes showed that the 24 genes are unevenly distributed on the 7 chromosomes, with 10 of them specifically located on chromosome 7H. A structure-based sequence alignment demonstrates that each XTH possesses a highly conserved domain (ExDxE) responsible for catalytic activity. Expression profiles based on the barley genome database showed that HvXTH family members display different expression patterns in different tissues and at different stages. This study is the first systematic genomic analysis of the barley HvXTH gene family. Our results provide valuable information that will help to elucidate the roles of HvXTH genes in the growth and development of barley.

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 resolubilise 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 0.5 h) 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.

Xyloglucan endotransglucosylase/hydrolase genes (XTHs) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum, Nicotiana sylvestrisis, Nicotiana tomentosiformis, Arabidopsis, and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

Highlight Expression of cell wall-related genes marks the onset of haustorium development in the parasitic plant Cuscuta. Action assays suggest a central role for xyloglucan endotransglucosylases/hydrolases in host plant infection., Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium.

Cellulose is an economically important material, but routes of its industrial processing have not been fully explored. The plant cell wall – the major source of cellulose – harbours enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family. This class of enzymes is unique in that it is capable of elongating polysaccharide chains without the requirement for activated nucleotide sugars (e.g., UDP-glucose) and in seamlessly splitting and reconnecting chains of xyloglucan, a naturally occurring soluble analogue of cellulose. Here, we show that a recombinant version of AtXTH3, a thus far uncharacterized member of the Arabidopsis XTH family, catalysed the transglycosylation between cellulose and cello-oligosaccharide, between cellulose and xyloglucan-oligosaccharide, and between xyloglucan and xyloglucan-oligosaccharide, with the highest reaction rate observed for the latter reaction. In addition, this enzyme formed cellulose-like insoluble material from a soluble cello-oligosaccharide in the absence of additional substrates. This newly found activity (designated “cellulose endotransglucosylase,” or CET) can potentially be involved in the formation of covalent linkages between cellulose microfibrils in the plant cell wall. It can also comprise a new route of industrial cellulose functionalization.

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.

Cadmium (Cd2+) is a toxic heavy metal impairing plant growth and development. Xyloglucan endotransglucosylase/hydrolase gene (XTH) is involved in the plant response to heavy metal toxicity, in addition to controlling cell wall extensibility. However, the link between XTH and Cd2+ stress has not yet been established in higher plants. PeXTH expression was up-regulated by 1.2–2.1-fold in Populus euphratica roots and leaves upon Cd2+ exposure (40–80 μM CdCl2). Cellular Cd2+ analysis and flux data showed that the cadmium-elicited expression of PeXTH markedly restricted Cd2+ uptake and accumulation in P. euphratica roots. Moreover, tobacco plants overexpressing PeXTH were more tolerant to Cd2+ stress (80 μM CdCl2) than wild-type tobacco in terms of root and shoot growth. Transgenic lines accumulated 49–58% less Cd2+ in root apical and mature regions, as compared to the wild type. The less buildup of Cd2+ in roots of transgenic lines was the result of lower influx of Cd2+ under Cd2+ stress. It is noting that transgenic plants displayed 56–87% higher xyloglucan degradation activity (XDA) than the wild type, leading to a 25–27% decline of xyloglucan content in the root cell walls. Therefore, overexpression of PeXTH increased the activity of XDA in transgenic plants, which enhanced the degradation of xyloglucan in the wall. The down-regulated amount of xyloglucan led to less binding sites for Cd2+ and thus reduced the root Cd2+ uptake and buildup in transgenic plants. Consequently, the Cd2+ toxicity was eventually alleviated in transgenic tobacco.

Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to contribute to fruit softening by degrading xyloglucan that is a predominant hemicellulose in the cell wall. In this study, two full-length XTH genes (DKXTH1 and DKXTH2) were identified from 'Fupingjianshi' persimmon fruit, and the expression level of both XTH genes was investigated during softening for 18-24 d using RT-qPCR. Sequence analysis showed that DKXTH1 and DKXTH2 contained a putative open reading frame of 861 and 876 bp encoding polypeptides of 287 and 292 amino acid residues, respectively, which contained the conserved DEIDFEFLG motif of XTH, a potential N-linked glycosylation signal site. RT-qPCR analysis showed that DKXTH1 and DKXTH2 in untreated fruit had different expression patterns during fruit softening, in which maximum expression occurred on days 3 and 12 of ripening, respectively. 1-Methylcyclopropene (1-MCP) and gibberellic acid (GA(3)) treatments delayed the softening and ethylene peak of persimmon fruit, as well as suppressed the expression of both XTH genes, especially DKXTH1. These results indicated that the expression of both XTH genes might be ethylene dependent action, and closely related to softening of persimmon in the early (DKXTH1) and later (DKXTH2) ripening stages.

DkXTH1 promoted cell elongation and more strength to maintain structural integrity by involving in cell wall assembly, thus enhanced tolerance to abiotic stress with broader phenotype in transgenic plants. Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to play a key role in cell wall modifications by cleaving and re-joining xyloglucan, and participates in the diverse physiological processes. DkXTH1 was found to peak in immature expanding persimmon fruit, and its higher expression level exhibited along with firmer fruit during storage. In the present study, transgenic Arabidopsis and tomato plants were generated with DkXTH1 constitutively expressed. Overexpression of DkXTH1 enhanced tolerance to salt, ABA and drought stresses in transgenic Arabidopsis plants with respect to root and leaf growth, and survival. Transgenic tomatoes collected at the mature green stage, presented delayed fruit softening coupled with postponed color change, a later and lower ethylene peak, and higher firmness in comparison with the wild-type tomatoes during storage. Furthermore, broader leaves and tomato fruit with larger diameter were gained in transgenic Arabidopsis and tomato, respectively. Most importantly, transgenic plants exhibited more large and irregular cells with higher density of cell wall and intercellular spaces, resulting from the overactivity of XET enzymes involving in cell wall assembly. We suggest that DkXTH1 expression resulted in cells with more strength and thickness to maintain structural integrity, and thus enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants.

Fruit softening is mainly associated with cell wall structural modifications, and members of the xyloglucan endotransglucosylase/hydrolase (XTH) family are key enzymes involved in cleaving and re-joining xyloglucan in the cell wall. In this work, we isolated a new XTH gene, DkXTH8, from persimmon fruit. Transcriptional profiling revealed that DkXTH8 peaked during dramatic fruit softening, and expression of DkXTH8 was stimulated by propylene and abscisic acid but suppressed by gibberellic acid and 1-MCP. Transient expression assays in onion epidermal cells indicated direct localization of DkXTH8 to the cell wall via its signal peptide. When expressed in vitro, the recombinant DkXTH8 protein exhibited strict xyloglucan endotransglycosylase activity, whereas no xyloglucan endohydrolase activity was observed. Furthermore, overexpression of DkXTH8 resulted in increased leaf senescence coupled with higher electrolyte leakage in Arabidopsis and faster fruit ripening and softening rates in tomato. Most importantly, transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring, resulting in wider interstitial spaces with less compact cells. We suggest that DkXTH8 expression causes cells to be easily destroyed, increases membrane permeability and cell peroxidation, and accelerates leaf senescence and fruit softening in transgenic plants.

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 of5000:1. 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.

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.

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.

Abscission is a process that involves shedding of plant organs from the main plant body. In this study it is shown that the process of petal separation in the fragrant rose, Rosa bourboniana, is accompanied by the expression of two xyloglucan endotransglucosylase/hydrolase genes, RbXTH1, and RbXTH2. The sequences of the two genes show 52% amino acid identity but are conserved at the catalytic site. The genes are up-regulated soon after the initiation of the abscission process and their transcription is associated with the progression of abscission, being faster in ethylene-treated flowers but slower during field abscission. Transcription is ethylene responsive, with the ethylene response being tissue-specific for RbXTH1 but largely tissue-independent for RbXTH2. Expression is correlated with an increase in xyloglucan endotransglucosylase (XET) action in petal abscission zones of both ethylene-treated and field abscising flowers. Proximal promoters of both the genes drive b-glucuronidase expression in an ethylene-responsive and abscission-related manner in agrobacteria-infiltrated rose petals, indicating that cis-elements governing ethylene-responsive and abscission-related expression probably lie within the first 700 nucleotides upstream of the translational initiation codon. The results show that cell wall remodelling of the xyloglucan moieties through the XET action of XTHs may be important for cell separation during abscission.

Some cell wall enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family catalyze molecular grafting between xyloglucan molecules. In tomato, 25 genes for XTH proteins have been identified. We studied the tomato gene SlXTH1, which has highest homology to the cDNA of the XTH first purified from hypocotyls of Vigna angularis. SlXTH1 mRNA was found to accumulate transiently at an early stage of fruit development, and the peak day of accumulation was about 12 days after pollination. The expression profile of SlXTH1 mRNA was roughly associated with that of the reported enzyme activity, suggesting that the SlXTH1 protein may be one of the main determinants of the enzyme activity. We then examined how alteration of expression of the SlXTH1 gene could influence the characteristics of fruits. Several lines of transgenic tomatoes with different levels of SlXTH1 transcript were produced by introducing a SlXTH1 transgene in either the sense or antisense orientation downstream of a constitutive promoter. In these transgenic tomatoes, fruit size was positively correlated with the level of SlXTH1 transcript, which was monitored at its peak stage. This is the first in vivo demonstration that SlXTH1 can control the morphological character of plants through changes in its level of expression.

While an increase in the number of xyloglucan tethers between the cellulose microfibrils in plant cell walls increases the walls' rigidity, the degradation of these tethers causes the walls to loosen. Degradation can occur either through the integration of xyloglucan oligosaccharides due to the action of xyloglucan endotransglucosylase or through direct hydrolysis due to the action of xyloglucanase. This is why the addition of xyloglucan and its fragment oligosaccharides causes plant tissue tension to increase and decrease so dramatically. Experiments involving the overexpression of xyloglucanase and cellulase have revealed the roles of xyloglucans in the walls. The degradation of wall xyloglucan in poplar by the transgenic expression of xyloglucanase, for example, not only accelerated stem elongation in the primary wall, but also blocked upright-stem gravitropism in the secondary wall. Overexpression of cellulase also reduced xyloglucan content in the walls as cellulose microfibrils were trimmed at their amorphous region, resulting in increased cell volume in Arabidopsis leaves and in sengon with disturbed leaf movements. The hemicellulose xyloglucan, in its function as a tether, plays a key role in the loosening and tightening of cellulose microfibrils: it enables the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.

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 [EC] ) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151 [EC] ) 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 (5.07.5), in temperature dependence and in acceptor substrate preferences.

The XTH Gene Family : An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Depolymerization of cell watt xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endo-transgtucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing watt-bound xyloglucan, or restructuring the existing cell watt material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SIXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SIXTHs contributed to the total XET and XEH activities. Our results showed that all of the SIXTHs studied were expressed during fruit growth and ripening, and that the expression of all. the SIXTHs in Group 1 was clearly related to fruit growth, as were SIXTH12 in Group 2 and SIXTH6 in Group 3-B. Only the expression of SIXTH5 and SIXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SIXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was tow and experienced a significant increase during fruit ripening, which was not correlated either with SIXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell watt, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes. (C) 2008 Elsevier GmbH. All rights reserved.

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 (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.

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH3 at 80 °C for 2–4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.

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.

In general, cell wall-degrading enzymes produced by plant pathogenic fungi are considered important pathogenicity factors. In this work, we evaluate the implication of xyloglucan endotransglucosylase/ hydrolase (XTHs), a potential hemicellulosic repairing enzyme, in the infection mechanism process by the fungus. This study investigated the SIXTHS expresion and xyloglucan endotransglucosylase (XET) activity during infection of two tomato fruit cultivars by Penicillium expansum Link. A. In infected fruits, XET specific activity decreased drastically after long infection periods, 24 and 48 h for Canario and Money Maker tomato fruits, respectively. Real Time RT-PCR of eleven SIXTHS also showed a decrease in expression as the infection progressed in both tomato fruit cultivars. Results suggest that the reduction in SIXTHS expression during infection might be related with the fungus attack mechanism. We suggest a possible transcriptional control of the SIXTHS expression by the fungus, causing a decrease in XET activity and, consequently, lower xyloglucan endotransglucosylation, which changes the xyloglucan structure. These changes might increase the fruit softening and wall disassembly, facilitating the fungus colonization and the progress of the infection.

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 microM. The 2-microM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Xyloglucan endotransglucosylase/hydrolases (XTHs) play roles in plant development by rearranging xyloglucan cross-links. Previously, we isolated a cDNA of XTH,VrXTH1, from mungbean that is thought to be associated with auxin-related growth. Here we report that several factors regulate the expression ofVrXTH1. This gene is predominantly expressed in the elongating regions of seedlings. Environmental stimuli, such as light and temperature, also affect its transcript levels. Because calcium acts as a second messenger in many signal transduction pathways, we examined its involvement in the regulation ofVrXTH1 expression. Interestingly, the application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; a specific calcium chelator) repressed transcription, regardless of the presence of auxin. Furthermore, treatment with either A23187 (a calcium ionophore) or calcium increased gene expression, suggesting that changes in the amount of intracellular Ca2+ is important to the modification of transcript levels. Taken together, our results demonstrate that the expression ofVrXTH1 is closely related to plant growth and may be modulated by the concentration of cytosolic calcium.

† Background and Aims In angiosperms xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is involved in reorganization of the cell wall during growth and development. The location of oligo-xyloglucan transglucosylation activity and the presence of XTH expressed sequence tags (ESTs) in the earliest diverging extant plants, i.e. in bryophytes and algae, down to the Phaeophyta was examined. The results provide information on the presence of an XET growth mechanism in bryophytes and algae and contribute to the understanding of the evolution of cell wall elongation in general. † Methods Representatives of the different plant lineages were pressed onto an XET test paper and assayed. XET or XET-related activity was visualized as the incorporation of fluorescent signal. The Physcomitrella genome database was screened for the presence of XTHs. In addition, using the 3 0 RACE technique searches were made for the presence of possible XTH ESTs in the Charophyta. † Key Results XET activity was found in the three major divisions of bryophytes at sites corresponding to growing regions. In the Physcomitrella genome two putative XTH-encoding cDNA sequences were identified that contain all domains crucial for XET activity. Furthermore, XET activity was located at the sites of growth in Chara (Charophyta) and Ulva (Chlorophyta) and a putative XTH ancestral enzyme in Chara was identified. No XET activity was identified in the Rhodophyta or Phaeophyta. † Conclusions XET activity was shown to be present in all major groups of green plants. These data suggest that an XET-related growth mechanism originated before the evolutionary divergence of the Chlorobionta and open new insights in the evolution of the mechanisms of primary cell wall expansion.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall-modifying enzymes that align within three or four distinct phylogenetic subgroups. One explanation for this grouping is association with different enzymic modes of action, as XTHs can have xyloglucan endotransglucosylase (XET) or endohydrolase (XEH) activities. While Group 1 and 2 XTHs predominantly exhibit XET activity, to date the activity of only one member of Group 3 has been reported: nasturtium TmXH1, which has a highly specialized function and hydrolyses seed-storage xyloglucan rather than modifying cell wall structure. Tomato fruit ripening was selected as a model to test the hypothesis that preferential XEH activity might be a defining characteristic of Group 3 XTHs, which would be expressed during processes where net xyloglucan depolymerization occurs. Database searches identified 25 tomato XTHs, and one gene (SlXTH5) was of particular interest as it aligned within Group 3 and was expressed abundantly during ripening. Recombinant SlXTH5 protein acted primarily as a transglucosylase in vitro and depolymerized xyloglucan more rapidly in the presence than in the absence of xyloglucan oligosaccharides (XGOs), indicative of XET activity. Thus, there is no correlation between the XTH phylogenetic grouping and the preferential enzymic activities (XET or XEH) of the proteins in those groups. Similar analyses of SlXTH2, a Group 2 tomato XTH, and nasturtium seed TmXTH1 revealed a spectrum of modes of action, suggesting that all XTHs have the capacity to function in both modes. The biomechanical properties of plant walls were unaffected by incubation with SlXTH5, with or without XGOs, suggesting that XTHs do not represent primary cell wall-loosening agents. The possible roles of SlXTH5 in vivo are discussed.

The plant cell wall is the structural basis of cellular form and thus forms a foundation on which morphogenesis builds organs and tissues. Enzymes capable of modifying major wall components are prominent candidates for regulating wall form and function. Xyloglucan endotransglucosylases/hydrolases (XTHs) are predicted to participate in xyloglucan integration and/or restructuring. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain insight into the potential physiological relevance of the distinct members of this family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data reveal that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs show XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes. These data suggest that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics.

• Background and Aims Oxaziclomefone (OAC), a new herbicide, inhibits cell expansion, especially in roots and cell-cultures of gramineous monocots. OAC does not affect turgor in cultured maize cells, and must therefore inhibit wall-loosening or promote wall-tightening. • Methods The effects of OAC in living cultured maize cells on various biochemical processes thought to influence wall extension were studied. • Key Results OAC did not affect 14C-incorporation from d-[U-14C]glucose into the major sugar residues of the cell wall (cellulosic glucose, non-cellulosic glucose, arabinose, xylose, galactose, mannose or uronic acids). OAC had no effect on 14C-incorporation from trans-[U-14C]cinnamate into wall-bound ferulate or its oxidative coupling-products. OAC did not influence the secretion or in-vivo action of peroxidase or xyloglucan endotransglucosylase activities—proposed wall-tightening and -loosening activities, respectively. The herbicide did not affect the consumption of extracellular l-ascorbate, an apoplastic solute proposed to act as an antioxidant and/or to generate wall-loosening hydroxyl radicals. • Conclusions OAC decreased wall extensibility without influencing the synthesis or post-synthetic modification of major architectural wall components, or the redox environment of the apoplast. The possible value of OAC as a probe to explore aspects of primary cell wall physiology is discussed.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are thought to be involved in various aspects of plant development by modifying the structure of xyloglucan cross-links. To address one of the roles of XTHs in plant growth, we identified an XTH, VrXTH1, in the mungbean through a differential reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of VrXTH1 shows high similarity to other XTHs. In addition, a signal peptide consisting of 17 amino acids is found at the N-terminus. The gene expression of VrXTH1 was differentially regulated in tissues and was higher in hypocotyls and stems than it was in other tissues. The steady state level of VrXTH1 transcripts was closely related to the elongation regions of hypocotyls. Notably, in the elongation region of hypocotyls, most VrXTH1 mRNAs were limited to the epidermis and to some layers of the cortex that act as growth-limiting tissue. Growth-promoting hormones, such as auxin and brassinolide, strongly enhanced mRNA accumulation of VrXTH1. However, abscisic acid, a hormone which is antagonistic to auxin, acted as a downregulator. Overall, VrXTH1 seems to play a role in plant growth at the gene level and, thus, by possibly altering cell wall morphogenesis in mungbean hypocotyls.