Xyloglucan Endotransglucosylase/hydrolase Research Articles

Function of xyloglucan endotransglucosylase/hydrolases in rice

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. 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. 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. 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.

Changes in xyloglucan endotransglucosylase/hydrolase (XTHs) expression and XET activity during apple fruit infection by Penicillium expansum Link. A

Although the plant cell wall is an extremely effective physical barrier against attack by pathogens, most phytopathogenic microorganisms produce enzymes that are capable of degrading cell wall polymers such as pectin and hemicelluloses. In this work we investigate the possible implication of the cell wall enzyme XET in the host-pathogen interaction as well as the XTH gene regulation during apple fruit infection by Penicillium expansum Link. A. We characterized the time course of fungal infection in apple fruit, and the XET activity and the expression of eleven MdXTHs. The results showed an important decrease in XET specific activity in the fruits after 24, 48 and 72 h of fruit infection (42.9 %, 73.1 % and 95.1 % respectively as compared with the controls). The expression analysis of the MdXTHs showed an important decrease in expression, as the infection progressed, in MdXTH2 and MdXTH10, as well as in MdXTH3, MdXTH4 and MdXTH5, but mainly after 72 h of fruit infection; and in MdXTH1, and MdXTH9 after 24 and 48 h of infection. These results suggested that the decrease in XET activity during infection could be mainly attributed to the decrease in expression of MdXTH2 and MdXTH10, since those were the MdXTHs that showed the highest expression levels during ripening, particularly MdXTH10. These results support the suggestion that inhibition of MdXTHs expression, and consequently the decrease in XET specific activity, could represent a pathogenicity factor, since the potential reconstructing role of the enzyme also decreased and the infection could progress.

Flower senescence: some molecular aspects

Some molecular aspects of flower senescence have been reviewed. The isolation, identification and characterization of different genes from various flowers (mainly from petals) associated with senescence have been discussed. The isolated genes were divided into different groups. A large proportion of genes have been found to be upregulated during flower senescence while some genes were also found to be downregulated indicating that there exists a complex interplay between the expression patterns of various genes. The genes involved in petal expansion are found to be upregulated during normal flower development from anthesis to open flower stage, but XTH (Xyloglucan endotransglucosylase hydrolase) is found to be involved in petal expansion as well as abscission. Cysteine proteases or the genes encoding cysteine proteases (assigned a central role in protein degradation) have been identified from various flower systems, but no cysteine protease has been identified from senescing Mirabilis jalapa flowers. In addition to proteases, the genes encoding ubiquitin (exhibiting proteasomal degradation by 26S proteasomes) have also been identified suggesting the two alternate pathways for protein degradation. Genes encoding specific nucleases have also been identified, but they displayed an early increase in transcript abundance before the senescence symptoms become evident and characterize the involvement of PCD during flower senescence. A range of transcription factors are described and their possible role in flower senescence has been discussed. A detailed description of genes involved in ethylene synthesis and the components involved in ethylene signaling have been presented.

Ethylene-induced changes in lignification and cell wall-degrading enzymes in the roots of mungbean (Vigna radiata) sprouts

As an important regulator, ethylene inhibits root growth and development in plants. To determine the mechanism of ethylene on root elongation growth and lateral root formation, ethylene-induced lignification and cell wall-degrading enzymes in the roots of mungbean sprouts were tested. We initially observed that primary root elongation and lateral root numbers were inhibited, while lignin content was enhanced by ethephon (ETH). Cell wall remolding proteins, polygalacturonase (PG) and carboxymethyl cellulose (Cx) activities were reduced, but α-expansins and xyloglucan endotransglucosylases/hydrolases (XTH) were enhanced by ETH. The promotion in lignin production was correlated with changes in activities of key lignin biosynthesis enzymes and hydrogen peroxide (H2O2) content. These actions induced by ETH were altered via treatment with an ethylene perception antagonist (Ag+). We subsequently demonstrated that the role of endogenous ethylene in regulating root elongation growth and lateral root formation were correlated with lignification and cell wall-degrading enzymes, respectively. These results suggested that the ethylene-regulated inhibition of primary root elongation growth was caused by an increase in lignification that reinforced the cell wall and shortened root length, and the suppression of lateral root formation was linked to activities of PG, Cx, α-expansins and XTH.

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.

Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls

Growth and biomechanics of etiolated hypocotyls from Arabidopsis thaliana lines overexpressing xyloglucan endotransglucosylase/hydrolase AtXTH18, AtXTH19, AtXTH20, and PttXET16-34 were studied. Overexpression of AtXTH18, AtXTH19, and AtXTH20 stimulated growth of hypocotyls, while PttXET16-34 overexpression did not show this effect. In vitro extension of frozen/thawed hypocotyls measured by a constant-load extensiometer started from a high-amplitude initial deformation followed by a slow time-dependent creep. Creep of growing XTH-overexpressing (OE) hypocotyls was more linear in time compared with the wild type at pH 5.0, reflecting their higher potential for long-term extension. XTH-OE plants deposited 65-84% more cell wall material per hypocotyl cross-sectional area than wild-type plants. As a result, their wall stress under each external load was lower than in the wild-type. Growing XTH-OE hypocotyls had higher values of initial deformation·stress(-1) compared with the wild type. Plotting creep rates for each line under different loads against the respective wall stress values gave straight lines. Their slopes and intercepts with the abscissa correspond to ϕ (in vitro cell wall extensibility) and y (in vitro cell wall yield threshold) values characterizing cell wall material properties. The wall material in XTH-OE lines was more pliant than in the wild type due to lower y values. In contrast, the acid-induced wall extension in vitro resulted from increasing ϕ values. Thus, three factors contributed to the XTH-OE-stimulated growth in Arabidopsis hypocotyls: their more linear creep, higher values of initial deformation·stress(-1), and lower y values.

Identification of xyloglucan endotransglucosylase/hydrolase genes (XTHs) and their expression in persimmon fruit as influenced by 1-methylcyclopropene and gibberellic acid during storage at ambient temperature

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 18–24d using RT-qPCR. Sequence analysis showed that DKXTH1 and DKXTH2 contained a putative open reading frame of 861 and 876bp 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 (GA3) 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.

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

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

Comparative proteomic analysis of pistil abortion in Japanese apricot (Prunus mume Sieb. et Zucc)

The phenomenon of pistil abortion widely occurs in Japanese apricot and has seriously affected the yield in production. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identify the differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot. More than 400 highly reproducible protein spots (P<0.05) were detected and 27 protein spots showed a greater than two-fold difference in their expression values. The proteins identified were classified into eight functional classifications and ten process categories, according to the Gene Ontology (GO). Acetyl-CoA produced by ATP citrate lyase (ACL) as a structural substance during formation of the cell wall could regulate pistil abortion in Japanese apricot. S-adenosylmethionine (SAM), xyloglucan endotransglucosylase/hydrolases (XTHs) and caffeoyl-CoA-O-methyl transferase (CCoAOMT) could promote cell wall formation in perfect flower buds of Japanese apricot, greatly contributing to pistil development. Spermidine hydroxycinnamoyl transferase (SHT) may be involved in the O-methylation of spermidine conjugates and could contribute to abnormal floral development. The identification of such differentially expressed proteins provides new targets for future studies that will assess their physiological roles and significance in pistil abortion.

Identification of desiccation-regulated genes by cDNA-AFLP in Haberlea rhodopensis: a resurrection plant

The Balkan endemic plant species Haberlea rhodopensis belongs to the group of resurrection plants. The members of this small group of angiosperms tolerate extreme dehydration of their vegetative tissues, which are able to recover very rapidly upon rehydration. In this respect, resurrection plants are a unique model intensively studied to reveal the secrets of desiccation tolerance. To date, the knowledge on the molecular biology of H. rhodopensis is very limited. Here, we report on the application of a cDNA-AFLP analysis to examine gene expression in leaves of Haberlea during dehydration. Twenty transcripts among 33 sequenced cDNA fragments appear to be involved in energy metabolism, transport, cell-wall biogenesis, signal transduction, or are probably transcription regulators according to their putative function. Expression patterns of two up-regulated (HrhDR8, HrhDR35) and two down-regulated (HrhDR6, HrhDR25) transcripts were verified by sqRT-PCR analysis at different stages of water stress. The results demonstrated that two up-regulated transcripts HrhDR8 and HrhDR35 encoding putative succinate-dehydrogenase and xyloglucan endotransglucosylase/hydrolase (XTH), respectively, were induced during early stage of dehydration, persist in desiccated state, and subsequent rehydration of Haberlea. Their possible involvement in drought tolerance is discussed.

Blue‐light‐mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.

Expression of ripening-related genes in cold-stored tomato fruit

The effects of chilling on fruit ripening and the expression of ripening-related genes have been investigated in M82IL2-2, a wild species introgression breeding tomato line ( Solanum lycopersicum × Solanum pennellii). Fruit harvested at the breaker stage of ripening were stored at 3 °C for 0, 1, 2 and 4 weeks, followed by 20 °C for 0–14 d. Fruit stored for 1 week ripened normally, as assessed by red color development and softening at 20 °C, but those stored for 2 or 4 weeks showed delayed or inhibited ripening. The concentrations of the carotenoids, phytoene, phytofluene, zeta (ζ)-carotene, gamma (γ)-carotene and lycopene, but not lutein and β-carotene, were reduced in chilled fruit. Microarray analysis showed that after storage at 3 °C for 4 weeks, 352 genes were up-regulated by chilling, whereas 321 genes were down-regulated, while after 7 d at 20 °C, 180 and 126 genes, respectively, were up- and down-regulated in chilled fruit. Chilling-induced changes included expression of transcriptional repressors such as a C2H2-type zinc finger protein. Expression of genes involved in color development, including phytoene synthase1 ( PSY1), carotenoid isomerase ( CRTISO), geranylgeranyl diphosphate synthase 2 ( GGPPS2), and 1-deoxy- d-xylulose-5-phosphate synthase ( DXS), showed reduced expression during and after chilling, as did genes encoding the cell wall modifying proteins polygalacturonase (PG), pectin esterase1 (PE1), β-galactosidase ( TBG4), expansin1 ( LeExp1), and xyloglucan endotransglucosylase-hydrolase 5 ( XTH5). Alcohol dehydrogenase 2 ( ADH2) and alcohol acyltransferase ( AAT) gene expression was also reduced by chilling. Alteration of ethylene production correlated with the altered ACC synthases ( ACS2, ACS4), and ACC oxidase ( ACO1) expression. The expression of genes involved in the ethylene signal transduction pathway, such as LeETR1, NR, LeETR4, LeCTR1, LeEIL3, LeEIL4, and LeERF3, was altered by chilling, suggesting that ethylene perception and sensitivity were affected. Chilling also reduced gene expression of a ripening-regulated transcription factor, LeMADS-RIN. The effect of chilling on ethylene biosynthesis, ethylene perception, the expression of a transcription factor necessary for ripening, and transcriptional repressors may contribute to the alteration of fruit ripening in tomato.

Alteration of fruit characteristics in transgenic tomatoes with modified expression of a xyloglucan endotransglucosylase/hydrolase gene

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.

陸上植物におけるエンド型キシログルカン転移酵素／加水分解酵素（XTH）の機能解析

陸上植物におけるエンド型キシログルカン転移酵素／加水分解酵素（XTH）の機能解析

Cell wall modification involving XTHs controls phytochrome-mediated petiole elongation in Arabidopsis thaliana

The shade avoidance syndrome serves to improve the competitive power of plants growing in crowded plant communities. An important element of avoiding shade is to rapidly elongate shoots and outgrow competing neighbours. We investigated the role of cell wall modifying proteins expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) in mediating this vital elongation growth in Arabidopsis thaliana. These proteins act on the cell wall and modify it to make it more extensible thereby facilitating cellular expansion. We found that XTHs are essential for shade-induced growth in Arabidopsis. Expansin activity on the other hand was not regulated in plants exposed to shade. Shade also resulted in rapid apoplastic acidification which is necessary for the optimal activity of cell wall modifying proteins such as XTHs and expansins.

Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening

Growth of petal cells is a basis for expansion and morphogenesis (outward bending) of petals during opening of carnation flowers (Dianthus caryophyllus L.). Petal growth progressed through elongation in the early stage, expansion with outward bending in the middle stage, and expansion of the whole area in the late stage of flower opening. In the present study, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) (DcXTH1–DcXTH4) and three cDNAs encoding expansin (DcEXPA1–DcEXPA3) were cloned from petals of opening carnation flowers and characterized. Real-time reverse transcription-PCR analyses showed that transcript levels of XTH and expansin genes accumulated differently in floral and vegetative tissues of carnation plants with opening flowers, indicating regulated expression of these genes. DcXTH2 and DcXTH3 transcripts were detected in large quantities in petals as compared with other tissues. DcEXPA1 and DcEXPA2 transcripts were markedly accumulated in petals of opening flowers. The action of XTH in growing petal tissues was confirmed by in situ staining of xyloglucan endotransglucosylase (XET) activity using a rhodamine-labelled xyloglucan nonasaccharide as a substrate. Based on the present findings, it is suggested that two XTH genes (DcXTH2 and DcXTH3) and two expansin genes (DcEXPA1 and DcEXPA2) are associated with petal growth and development during carnation flower opening.

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.

Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana

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) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) 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 (5.0-7.5), in temperature dependence and in acceptor substrate preferences.

Light quality-mediated petiole elongation in Arabidopsis during shade avoidance involves cell wall modification by xyloglucan endotransglucosylase/hydrolases.

Some plants can avoid shaded conditions via rapid shoot elongation, thus growing into better lit areas in a canopy. Cell wall-modifying mechanisms promoting this elongation response, therefore, are important regulatory points during shade avoidance. Two major cell wall-modifying protein families are expansins and xyloglucan endotransglucosylase/hydrolases (XTHs). The role of these proteins during shade avoidance was studied in Arabidopsis (Arabidopsis thaliana). In response to two shade cues, low red to far-red light (implying neighbor proximity) and green shade (mimicking dense canopy conditions), Arabidopsis showed classic shade avoidance features: petiole elongation and leaf hyponasty. Measurement of the apoplastic proton flux in green shade-treated petioles revealed a rapid efflux of protons into the apoplast within minutes, unlike white light controls. This apoplastic acidification probably provides the acidic pH required for the optimal activity of cell wall-modifying proteins like expansins and XTHs. Acid-induced extension, expansin susceptibility, and extractable expansin activity were similar in petioles from white light- and shade-treated plants. XTH activity, however, was high in petioles exposed to shade treatments. Five XTH genes (XTH9, -15, -16, -17, and -19) were positively regulated by low red to far-red light conditions, while the latter four and XTH22 showed a significant up-regulation also in response to green shade. Consistently, knockout mutants for two of these XTH genes also had reduced or absent shade avoidance responses to these light signals. These results point toward the cell wall as a vital regulatory point during shade avoidance.

Modification of cell wall properties in lettuce improves shelf life.

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.