Ride to cell wall: Arabidopsis XTH11, XTH29 and XTH33 exhibit different secretion pathways and responses to heat and drought stress

The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan–cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR-truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real-time polymerase chain reaction analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS-secreted proteins.     

A xyloglucan endotransglucosylase/hydrolase involves in growth of primary root and alters the deposition of cellulose in <Emphasis Type="Italic">Arabidopsis</Emphasis>

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.     

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. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

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.     

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

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

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

The AtXTH28 Gene, a Xyloglucan Endotransglucosylase/Hydrolase, is Involved in Automatic Self-Pollination in Arabidopsis thaliana

Successful automatic self-pollination in flowering plants isdependent on the correct development of reproductive organs.In the stamen, the appropriate growth of the filament, whichlargely depends on the mechanical properties of the cell wall,is required to position the anther correctly close to the stigmaat the pollination stage. Xyloglucan endotransglucosylase/hydrolases(XTHs) are a family of enzymes that mediate the constructionand restructuring of xyloglucan cross-links, thereby controllingthe extensibility or mechanical properties of the cell wallin a wide variety of plant tissues. Our reverse genetic analysishas revealed that a loss-of-function mutation of an ArabidopsisXTH family gene, AtXTH28, led to a decrease in capability forself-pollination, probably due to inhibition of stamen filamentgrowth. Our results also suggest that the role of AtXTH28 inthe development of the stamen is not functionally redundantwith its closest paralog, AtXTH27. Thus, our finding indicatesthat AtXTH28 is specifically involved in the growth of stamenfilaments, and is required for successful automatic self-pollinationin certain flowers in Arabidopsis thaliana.     

Differential expression of AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes in Arabidopsis roots. Physiological roles in specification in cell wall construction

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that are capable of splitting and reconnecting xyloglucan molecules, and are implicated in the construction and restructuring of the cellulose/xyloglucan framework. Thirty-three members of the XTH gene family are found in the genome of Arabidopsis thaliana, but their roles remain unclear. Here, we describe the tissue-specific and growth stage-dependent expression profiles of promoter::GUS fusion constructs for four Arabidopsis XTH genes, AtXTH17, AtXTH18, AtXTH19 and AtXTH20, which are phylogenetically closely related to one another. AtXTH17 and AtXTH18 were expressed in all cell types in the elongating and differentiating region of the root, while AtXTH19 was expressed in the apical dividing and elongating regions, as well as in the differentiation zone, and was up-regulated by auxin. In contrast, AtXTH20 was expressed specifically in vascular tissues in the basal mature region of the root. This expression analysis also disclosed cis-regulatory sequences that are conserved among the four genes, and are responsible for the root-specific expression profile. These results indicate that the four XTH genes, which were generated by gene duplication, have diversified their expression profile within the root in such a way as to take responsibility for particular physiological roles in the cell wall dynamics.     

Evolution and divergence in the coding and promoter regions of the Populus gene family encoding xyloglucan endotransglycosylase/hydrolases

Xyloglucan endotransglycosylase/hydrolases (XTHs) are believed to modify the cell wall structure by cleaving a xyloglucan polymer and transferring the newly generated, potentially reducing, terminal to another xyloglucan. We report here the detailed analysis of 37 Populus trichocarpa XTH genes/proteins in their divergence in both the coding and 5′ promoter regions. Our results show that the Populus XTH genes have experienced whole-genome and local duplications and pre- and post-speciation divergence. Genome-wide and segmental duplications seem to be dominant in subfamily I and III, while tandem duplication seems to be the major mechanism for the subfamily II expansion, which also has higher average ratios of K _a/K _s compared to those in subfamily I and III. There was a general lack of organ-specific gene expression. In contrast, the expression patterns in subfamily II varied in response to various hormone treatments, with II-A being up-regulated and II-B down-regulated after 2 h of hormone treatment. Expression for this subfamily was verified using the 1.5-kb PtXTH22 promoter that was fused with the GUS reporter gene and transformed into Arabidopsis. The PtXTH22 promoter contains auxin response element, ethylene insensitive 3-like factors, and brassinosteroid response cis-elements. Histochemical GUS staining of transgenic Arabidopsis seedlings confirmed that the PtXTH22 promoter was up-regulated by several hormones.     

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207and/or EC 3.2.1.15 [EC] 1) are enzymes involved in the modificationof cell wall structure by cleaving and, often, also re-joiningxyloglucan molecules in primary plant cell walls. Using a poolof antibodies raised against an enriched cell wall protein fraction,a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNAexpression library obtained from the elongation zone of themaize root. The predicted protein has a putative N-terminalsignal peptide and possesses the typical domains of this enzymefamily, such as a catalytic domain that is homologous to thatof Bacillus macerans β-glucanase, a putative N-glycosylationmotif, and four cysteine residues in the central and C terminalregions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1reveals that it belongs to subgroup 4, so far only reportedfrom Poaceae monocot species. ZmXTH1 has been expressed in Pichiapastoris (a methylotrophic yeast) and the recombinant enzymeshowed xyloglucan endotransglucosylase but not xyloglucan endohydrolaseactivity, representing the first enzyme belonging to subgroup4 characterized in maize so far. Expression data indicate thatZmXTH1 is expressed in elongating tissues, modulated by cultureconditions, and induced by gibberellins. Transient expressionassays in onion cells reveal that ZmXTH1 is directed to thecell wall, although weakly bound. Finally, Arabidopsis thalianaplants expressing ZmXTH1 show slightly increased xyloglucanendohydrolase activity and alterations in the cell wall structureand composition. Key words: Cell elongation, cell wall, plant transformation, XEH, XET, XTH, Zea mays     

Xyloglucan endotransglucosylase activity loosens a plant cell wall

BACKGROUND AND AIMS: 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. METHODS: 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. KEY RESULTS: 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. CONCLUSIONS: The results provide evidence that XTHs can act as cell wall-loosening enzymes.     

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

Tamarind xyloglucan was oxidised by reaction with sodium hypochlorite in the presence of 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO). Galactose residues and non-xylosylated glucose residues were thus converted into galacturonic and glucuronic acid residues, respectively, producing an anionic polysaccharide. Acid hydrolysis of oxidised xyloglucan yielded two aldobiouronic acids, deduced to be β-d-GalpA-(1→2)-d-Xyl and β-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)-β-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.     

Function of xyloglucan endotransglucosylase/hydrolases in rice

Background and Aims

Although xyloglucans are ubiquitous in land plants, they are less abundant in Poales species than in eudicotyledons. Poales cell walls contain higher levels of β-1,3/1,4 mixed-linked glucans and arabinoxylans than xyloglucans. Despite the relatively low level of xyloglucans in Poales, the xyloglucan endotransglucosylase/hydrolase (XTH) gene family in rice (Oryza sativa) is comparable in size to that of the eudicotyledon Arabidopsis thaliana. This raises the question of whether xyloglucan is a substrate for rice XTH gene products, whose enzyme activity remains largely uncharacterized.

Methods

This study focused on OsXTH19 (which belongs to Group IIIA of the XTH family and is specifically expressed in growing tissues of rice shoots), and two other XTHs, OsXTH11 (Group I/II) and OsXTH20 (Group IIIA), for reference, and measurements were made of the enzymatic activities of three recombinant rice XTHs, i.e. OsXTH11, OsXTH20 and OsXTH19.

Key Results

All three OsXTH gene products have xyloglucan endohydrolase (XEH, EC 3·2·1·151) activity, and OsXTH11 has both XEH and xyloglucan endotransglycosylase (XET, EC 2·4·1207) activities. However, these proteins had neither hydrolase nor transglucosylase activity when glucuronoarabinoxylan or mixed-linkage glucan was used as the substrate. These results are consistent with histological observations demonstrating that pOsXTH19::GUS is expressed specifically in the vicinity of tissues where xyloglucan immunoreactivity is present. Transgenic rice lines over-expressing OsXTH19 (harbouring a Cauliflower Mosaic Virus 35S promoter::OsXTH19 cDNA construct) or with suppressed OsXTH19 expression (harbouring a pOsXTH19 RNAi construct) did not show dramatic phenotypic changes, suggesting functional redundancy and collaboration among XTH family members, as was observed in A. thaliana.

Conclusions

OsXTH20 and OsXTH19 act as hydrolases exclusively on xyloglucan, while OsXTH11 exhibits both hydrolase and XET activities exclusively on xyloglucans. Phenotypic analysis of transgenic lines with altered expression of OsXTH19 suggests that OsXTH19 and related XTH(s) play redundant roles in rice growth.