Xyloglucan Endotransglucosylase/hydrolase Research Articles

Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage beta-glucan. Differences were found in pH- and temperature-dependence as well as in acceptor-substrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.

Re-interpreting the role of endo-β-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall

Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-beta-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-beta-mannanases to be reinterpreted. In this review, the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported. There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.

The AtXTH28 gene, a xyloglucan endotransglucosylase/hydrolase, is involved in automatic self-pollination in Arabidopsis thaliana.

Successful automatic self-pollination in flowering plants is dependent on the correct development of reproductive organs. In the stamen, the appropriate growth of the filament, which largely depends on the mechanical properties of the cell wall, is required to position the anther correctly close to the stigma at the pollination stage. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of xyloglucan cross-links, thereby controlling the extensibility or mechanical properties of the cell wall in a wide variety of plant tissues. Our reverse genetic analysis has revealed that a loss-of-function mutation of an Arabidopsis XTH family gene, AtXTH28, led to a decrease in capability for self-pollination, probably due to inhibition of stamen filament growth. Our results also suggest that the role of AtXTH28 in the development of the stamen is not functionally redundant with its closest paralog, AtXTH27. Thus, our finding indicates that AtXTH28 is specifically involved in the growth of stamen filaments, and is required for successful automatic self-pollination in certain flowers in Arabidopsis thaliana.

A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

BackgroundCell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.ResultsTwo developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.ConclusionThis work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

Xyloglucan endotransglucosylase/hydrolases (XTHs) during tomato fruit growth and ripening

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/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 wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs 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 SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs 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 low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH 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 wall, 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.

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.

Xyloglucan Endotransglucosylase Activity Loosens a Plant Cell Wall

Plant cells undergo cell expansion when a temporary imbalance between the hydraulic pressure of the vacuole and the extensibility of the cell wall makes the cell volume increase dramatically. The primary cell walls of most seed plants consist of cellulose microfibrils tethered mainly by xyloglucans and embedded in a highly hydrated pectin matrix. During cell expansion the wall stress is decreased by the highly controlled rearrangement of the load-bearing tethers in the wall so that the microfibrils can move relative to each other. Here the effect was studied of a purified recombinant xyloglucan endotransglucosylase/hydrolase (XTH) on the extension of isolated cell walls. The epidermis of growing onion (Allium cepa) bulb scales is a one-cell-thick model tissue that is structurally and mechanically highly anisotropic. In constant load experiments, the effect of purified recombinant XTH proteins of Selaginella kraussiana on the extension of isolated onion epidermis was recorded. Fluorescent xyloglucan endotransglucosylase (XET) assays demonstrate that exogeneous XTH can act on isolated onion epidermis cell walls. Furthermore, cell wall extension was significantly increased upon addition of XTH to the isolated epidermis, but only transverse to the net orientation of cellulose microfibrils. The results provide evidence that XTHs can act as cell wall-loosening enzymes.

The Implication of Xyloglucan Endotransglucosylase/Hydrolase (XTHs) in Tomato Fruit Infection by Penicillium expansum Link. A

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 SlXTHs 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 SlXTHs also showed a decrease in expression as the infection progressed in both tomato fruit cultivars. Results suggest that the reduction in SlXTHs expression during infection might be related with the fungus attack mechanism. We suggest a possible transcriptional control of the SlXTHs 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.

Gene expression profiles for cell wall-modifying proteins associated with soybean cyst nematode infection, petiole abscission, root tips, flowers, apical buds, and leaves

Changes in transcript accumulation for cell wall-modifying proteins were examined in excised soybean root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-beta-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3' and 5' RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean roots inoculated with SCN, non-inoculated roots, serial dissections of root tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized root pieces. Gene expression in serial dissections of root tips (0-2 mm, and 2-7 mm) and whole roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in roots. Also of interest, many of the cDNAs examined were up-regulated in petiole abscission zones induced to abscise with ethylene.

The α- and β-expansin and xyloglucan endotransglucosylase/hydrolase gene families of wheat: Molecular cloning, gene expression, and EST data mining

Expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) are families of extracellular proteins with members that have been shown to play an important role in cell wall growth. In this study, three, six, and five members of the wheat α-expansin ( TaEXPA1 to TaEXPA3), β-expansin ( TaEXPB1 to TaEXPB6), and XTH ( TaXTH1 to TaXTH5) gene families, respectively, were isolated from a dwarf wheat line. The mRNA expression analysis by real-time RT-PCR indicates that these genes display different transcription levels in different stages/organs/treatments, possibly suggesting their functional roles in the cell wall expansion process. Moreover, the comparison of the expression levels reveals that most of the expansins show lower expression than the XTHs. Finally, we present the analysis of wheat α- and β-expansins and XTH families by expressed sequence tag data mining.

A xyloglucan endotransglucosylase/hydrolase involves in growth of primary root and alters the deposition of cellulose in Arabidopsis

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that mediate the construction and restructure of the cellulose/xyloglucan framework by splitting and reconnecting xyloglucan molecule cross-linking among cellulose microfibrils. Remodification of cellulose microfibrils within cell-wall matrices is realized to be one of the most critical steps in the regulation of cells expansion in plants. Thirty-three XTH genes have been found in Arabidopsis thaliana but their roles remain unclear. AtXTH21 (At2g18800), an Arabidopsis XTH gene that mainly expresses in root and flower, exhibits different expression profiles from other XTH members under hormone treatment. We examined loss-of-function mutants using T-DNA insertion lines and overexpression lines and found that the AtXTH21 gene played a principal role in the growth of the primary roots by altering the deposition of cellulose and the elongation of cell wall.

Effects of hypergravity on expression of XTH genes in azuki bean epicotyls

Hypergravity produced by centrifugation caused inhibition of elongation growth and a decrease in the cell wall extensibility in azuki bean epicotyls (Vigna angularis Ohwi et Ohashi). Also, hypergravity increased the molecular mass of xyloglucans, whereas it decreased xyloglucan-degrading activity in epicotyls. When the expression profiles of three xyloglucan endotransglucosylase/hydrolase (XTH) genes, VaXTHS4, VaXTH1 and VaXTH2, were analyzed under hypergravity conditions, the expression of VaXTHS4, which shows only hydrolase activity, was downregulated in proportion to the logarithm of the magnitude of gravity (R = -0.94). However, the gene expression of VaXTH1 or VaXTH2, which shows only transglucosylase activity, was not affected by gravitational conditions. When the seedlings that had been grown at 1 g were transferred to hypergravity conditions at 300 g, the downregulation of VaXTHS4 expression was detected within 1 h. By removal of hypergravity stimulus, VaXTHS4 expression was increased within 1 h. These results suggest that azuki bean epicotyls promptly regulate the expression level of only VaXTHS4 in response to gravity stimuli. The regulation of xyloglucan-hydrolyzing activity as a result of changes in VaXTHS4 expression may be involved in the regulation by gravity of molecular mass of xyloglucans, leading to modifications of cell wall mechanical properties and cell elongation. Lanthanum and gadolinium, potential blockers of mechanosensitive calcium ion permeable channels (mechanoreceptors), nullified the suppression of VaXTHS4 expression, suggesting that mechanoreceptors are responsible for inhibition by hypergravity of VaXTHS4 expression.

Regulation ofVrXTH1 expression in mungbean

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.

シロイヌナズナのエンド型キシログルカン転移酵素/加水分解酵素(XTH)遺伝子ファミリー クラスIIIに属するメンバーの発現と機能の網羅的解析

シロイヌナズナのエンド型キシログルカン転移酵素/加水分解酵素(XTH)遺伝子ファミリー クラスIIIに属するメンバーの発現と機能の網羅的解析

XET activity is found near sites of growth and cell elongation in bryophytes and some green algae: new insights into the evolution of primary cell wall elongation.

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. 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' RACE technique searches were made for the presence of possible XTH ESTs in the Charophyta. 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. 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.

Why are Chloris gayana leaves shorter in salt-affected plants? Analyses in the elongation zone

Reduced hydraulic conductance calculated from growth data was suggested to be the main reason for reduced leaf expansion in salt-stressed Chloris gayana (Rhodes grass). In this work, xylem vessel cross-sections and wall enzyme activities were analysed to re-examine the effects of salinity on leaf growth in this species. Maximal segmental growth rates were 20% lower and the growth zone was 23% shorter in leaves from salinized plants than in controls; however, growth rates between 0 mm and 15 mm from the ligule were similar in both types of leaves. Xylem cross-sectional areas in this region were about 65% smaller in leaves of salinized plants, suggesting that hydraulic restrictions in the leaves of salinized plants were much higher than overall growth reductions. Extractable xyloglucan endotransglucosylase activity in this zone was twice as high in leaves of salinized plants as in leaves of controls. Nevertheless, the activity of the extracted enzyme was not affected by up to 1 M NaCl added to the reaction medium. Therefore, increased xyloglucan endotransglucosylase activity under salinity may be due to a promotion of transcription of XTH (xyloglucan endotransglucosylase/hydrolases) genes and/or translation of preformed transcripts. These results suggest that, as in drought stress, increased activity of cell wall enzymes associated with wall loosening may contribute to the maintenance of growth under saline conditions despite hydraulic restrictions.

Characterization of a new xyloglucan endotransglucosylase/hydrolase (XTH) from ripening tomato fruit and implications for the diverse modes of enzymic action

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.

Developmental Expression Patterns of Arabidopsis XTH Genes Reported by Transgenes and Genevestigator

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.