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.     

Mixed-linkage beta-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.     

Developmental expression of the cucumber <Emphasis Type="Italic">Cs-XTH1</Emphasis> and <Emphasis Type="Italic">Cs-XTH3</Emphasis> genes,encoding xyloglucan endotransglucosylase/hydrolases,can be influenced by mechanical stimuli

The expression of two genes encoding xyloglucan endotransglucosylase/hydrolases (XTHs), Cs-XTH1 and Cs-XTH3, was upregulated during the onset of cucumber somatic embryogenesis. As a means of characterising the developmental regulation of these genes, the activity of the respective upstream regulatory regions was investigated in seedlings and somatic embryos of Arabidopsis thaliana and Cucumis sativus. GUS assays revealed that both genes are under developmental control. In addition, elevated promoter activity was found in the tension-bearing regions of the plant and in response to touch and wounding, which is consistent with the existence of numerous stress-related cis elements in the 5′-regulatory regions. In vivo xyloglucan endotransglucosylase (XET) action assays were performed to gain an overview on the role of XTHs during somatic embryogenesis. The highest XET action was observed in the external cell layers of somatic embryos in the cotyledonary region and in the presumptive region of peg formation. Based on the results, we propose a dual mechanism (one developmental and the second adaptive) for the regulation of Cs-XTH1 and Cs-XTH3 activity wherein the developmental pattern can be modified by mechanical stimuli.     

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.     

Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

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 μM. The 2-μM 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.     

Nitric oxide exacerbates Al-induced inhibition of root elongation in rice bean by affecting cell wall and plasma membrane properties

Aluminum (Al) toxicity is one of the most widespread problems for crop production on acid soils, and nitric oxide (NO) is a key signaling molecule involved in the mediation of various biotic and abiotic stresses in plants. Here we found that exogenous application of the NO donor sodium nitroprusside (SNP) exacerbated the inhibition of Al-induced root growth in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi ‘Jiangnan’, Fabaceae]. This was accompanied by an increased accumulation of Al in the root apex. However, Al treatments had no effect on endogenous NO concentrations in root apices. These results indicate that a change in NO concentration is not the cause of Al-induced root growth inhibition and the adverse effect of SNP on Al-induced root growth inhibition should result from increased Al accumulation. Al could significantly induce citrate efflux but SNP had no effects on citrate efflux either in the absence or presence of Al. On the other hand, SNP pretreatment significantly increased Al-induced malondialdehyde accumulation and Evans Blue staining, indicating an intensification of the disruption of plasma membrane integrity. Furthermore, SNP pretreatment also caused greater induction of pectin methylesterase activity by Al, which could be the cause of the increased Al accumulation. Taken together, it is concluded that NO exacerbates Al-induced root growth inhibition by affecting cell wall and plasma membrane properties.     

In vivo colocalization of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of Arabidopsis roots

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO-SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO-SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide-SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO-SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.     

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation

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.     

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

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' 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 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.     

Involvement of putrescine and nitric oxide in aluminum tolerance by modulating citrate secretion from roots of red kidney bean

Background and Aims

Polyamines and nitric oxide (NO) are two important molecules modulating numerous environment stresses in plants. This study was to investigate the roles of polyamines and NO in aluminum (Al) tolerance in red kidney bean.

Methods

The interaction between putrescine (Put) and NO under Al stress was examined. NO donor and scavenger were used to further examine the role of NO in Al-induced citrate secretion from roots by high performance liquid chromatography.

Results

Al stress caused increase of endogenous free Put, and exogenous Put alleviated Al-induced inhibition of root elongation and Al accumulation. In addition, Put induced NO production and nitrate reductase (NR) activity under Al stress. Al- and Put-induced NO production could be reversed by NR inhibitor. Furthermore, Al stress stimulated citrate secretion from roots, and this response was stimulated by NO donor, whereas NO scavenger inhibited Al-induced citrate secretion from roots. Concomitantly, NO donor reduced Al accumulation in root apexes, while NO scavenger further enhanced Al accumulation. Al-induced inhibition of root growth was significantly improved by exogenous citrate treatment.

Conclusions

Put and NO enhanced Al tolerance by modulating citrate secretion from roots, and NO may act downstream of Put in red kidney bean under Al stress.     

Xyloglucan endotransglycosylase activity in pine hypocotyls. Intracellular localization and relationship with endogenous growth

Since xyloglucan depolymerization has been proposed as one of the biochemical bases for cell wall‐loosening in gymnosperms, we characterized xyloglucan endotransglycosylase (XET) activity during pine hypocotyl growth to establish a possible relationship. XET activity was measured as the incorporation of [³H]XXXGol into partially purified pine hypocotyl xyloglucan. XET specific and total activity was determined in the subapical and basal segments of pine hypocotyls at two different stages of growth in different subcellular fractions. XET activity was found in the apoplastic fluid, the symplastic fluid, and in the fraction of proteins ionically and covalently bound to the cell walls with different distribution profiles. The results showed a relationship between XET activity and hypocotyl growth in all the fractions, suggesting an important role for XET during growth. Consequently, the suggested growth‐promoting effect of XET in angiosperms can also be extended to gymnosperms. Also, the results demonstrate that XET bound to the cell wall is able to act on endogenous wall‐bound xyloglucan as well as soluble polymeric xyloglucan, using them as substrates for the endotransglycosylation reaction.     

Change in XET activities,cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential

In dark-grown soybean (Glycine max [L.] Merr.) seedlings, exposing the roots to water-deficient vermiculite (_w=–0.36 MPa) inhibited hypocotyl (stem) elongation. The inhibition was associated with decreased extensibility of the cell walls in the elongation zone. A detailed spatial analysis showed xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity on the basis of unit cell wall dry weight was decreased in the elongation region after seedlings were transplanted to low _w. The decrease in XET activity was at least partially due to an accumulation of cell wall mass. Since cell number was only slightly altered, wall mass had increased per cell and probably led to increased wall thickness and decreased cell wall extensibility. Alternatively, an increase in cell wall mass may represent a mechanism for regulating enzyme activity in cell walls, XET in this case, and therefore cell wall extensibility. Hypocotyl elongation was partially recovered after seedlings were grown in low-_w vermiculate for about 80 h. The partial recovery of hypocotyl elongation was associated with a partial recovery of cell wall extensibility and an enhancement of XET activity in the hypocotyl elongation zone. Our results indicate XTH proteins may play an important role in regulating cell wall extensibility and thus cell elongation in soybean hypocotyls. Our results also showed an imperfect correlation of spatial elongation and XET activity along the hypocotyls. Other potential functions of XTH and their regulation in soybean hypocotyl growth are discussed.     

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.     

Al Inhibits Both Shoot Development and Root Growth in als3, an Al-Sensitive Arabidopsis Mutant

In als3, an Al-sensitive Arabidopsis mutant, shoot development and root growth are sensitive to Al. Mutant als3 seedlings grown in an Al-containing medium exhibit severely inhibited leaf expansion and root growth. In the presence of Al, unexpanded leaves accumulate callose, an indicator of Al damage in roots. The possibility that the inhibition of shoot development in als3 is due to the hyperaccumulation of Al in this tissue was examined. However, it was found that the levels of Al that accumulated in shoots of als3 are not different from the wild type. The inhibition of shoot development in als3 is not a consequence of nonspecific damage to roots, because other metals (e.g. LaCl3 or CuSO4) that strongly inhibit root growth did not block shoot development in als3 seedlings. Al did not block leaf development in excised als3 shoots grown in an Al-containing medium, demonstrating that the Al-induced damage in als3 shoots was dependent on the presence of roots. This suggests that Al inhibition of als3 shoot development may be a delocalized response to Al-induced stresses in roots following Al exposure.