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.     

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.     

植物细胞壁松弛因子

植物细胞壁的松弛是细胞伸长必需的一个生理过程,发生于植物生长发育的各个阶段。研究发现参与细胞壁松弛的因子有多种,主要包括膨胀素（expansin）、木葡聚糖内转糖苷酶/水解酶（XTH）、糖基水解酶和羟基自由基（·OH）四大类。本文主要对这些细胞壁松弛因子的结构特征、作用机制及其在植物生理过程中的作用等方面的研究进展进行综述。     

BcXTH1, a Brassica campestris homologue of Arabidopsis XTH9, is associated with cell expansion

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of the enzymes that are responsible for reorganization of the cellulose–xyloglucan framework by catalyzing cleavage and religation of the xyloglucan chains in the plant cell wall. In this study, we report the isolation and characterization of a XTH gene from a pistil cDNA library of Brassica campestris. Sequence analysis of the gene, designated BcXTH1, revealed that it is homologous to the XTH9 gene of Arabidopsis. The highly conserved domain (DEIDFEFLG) found among all XTHs was also present in BcXTH1 but with the two amino acid substitutions (NEFDFEFLG) also found in Arabidopsis XTH9. These results suggest that BcXTH1 is the B. campestris homologue of XTH9. Expression analysis of BcXTH1 revealed that it was expressed in most of the plant organs. In situ hybridization showed that the gene is highly expressed in the floral primodia, especially in the epidermal cell layer. Southern blot analysis indicated that the BcXTH1 gene exists as a multi-copy gene in the B. campestris genome. The function of the BcXTH1 gene was deduced from using an overexpression strategy in Arabidopsis. Interestingly, the transgenic plants showed a pronounced cell expansion phenotype. Immunoelectron microscopy shows that BcXTH1 is localized almost exclusively to the cell wall, supporting our conclusion that it participates in the regulation of cell expansion in B. campestris.     

RNAi‐mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O‐methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down‐regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3′ direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down‐regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.     

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter‐connections of plant cell‐wall matrix glycans

Plant cell walls are complex, multi‐macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell‐wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell‐wall matrix glycan sub‐populations and inter‐connections. The method combines chromatographic separations with use of glycan‐directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell‐wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub‐populations and their modulation in A. thaliana organs.     

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.     

Oxaziclomefone, a new herbicide, inhibits wall expansion in maize cell-cultures without affecting polysaccharide biosynthesis, xyloglucan transglycosylation, peroxidase action or apoplastic ascorbate oxidation

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

Narrowing down the single homoeologous FaPFRU locus controlling flowering in cultivated octoploid strawberry using a selective mapping strategy

Extending the period of fruit production is a way to substantially increase crop yield in many fruit or ornamental species. In the cultivated octoploid strawberry (Fragaria × ananassa), the most consumed small fruit worldwide, fruit production season can be extended by selecting the perpetual flowering (PF) cultivars. This trait is of considerable interest to growers and to the food industry. Four homoeologous loci controlling a single trait can be expected in such a complex octoploid species. However, we recently showed that the PF trait is under the control of the single dominant FaPFRU locus (J. Exp. Bot., 2013, 64 , 1837), making it potentially amenable to marker‐assisted selection (MAS). Here, we report the successful use of a strategy, based on a selective mapping using a reduced sample of individuals, to identify nine markers in close linkage to the FaPFRU allelic variant. Thus, this strategy can be used to fine map the target homoeologous loci in other complex polyploid crop species. Recombinant analysis further enabled us to reduce the locus to a region flanked by two markers, Bx083_206 and Bx215_131, corresponding to a 1.1 Mb region in the diploid F. vesca reference genome. This region comprises 234 genes, including 15 flowering associated genes. Among these, the FLOWERING LOCUS T (FT) is known to be a key activator of flowering. The close association between the PF trait and the FaPFRU flanking markers was validated using an additional segregating population and genetic resources. This study lays the foundation for effective and rapid breeding of PF strawberry cultivars by MAS.     

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

The xyloglucan endotransglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodeling. The evolutionary history of plant XTH gene products is incompletely understood vis‐à‐vis the larger body of bacterial endoglycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high‐resolution X‐ray crystal structures and detailed enzyme kinetics of an extant transitional plant endoglucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)‐mixed‐linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed‐linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active‐site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endotranglycosylase (XET), and a xyloglucan endohydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species.     

Changes in the structure of xyloglucan during cell elongation

Xyloglucans were isolated by sequential extraction of the cell walls of pea (Pisum sativum L. cv. Alaska) with a xyloglucan-specific endoglucanase and KOH. The xyloglucan content and xyloglucan-oligosaccharide composition were determined for fractions obtained from the elongating and non-elongating segments of pea stems grown in the light and in darkness. The results were consistent with the hypothesis that regulated growth of the cell wall depends on xyloglucan metabolism. Furthermore, the characterization of xyloglucan extracted from leaves of light-grown pea plants indicates that xyloglucan metabolism is tissue specific. Changes in xyloglucan subunit structure observed in elongating stems are consistent with the in muro realization of a metabolic pathway that was previously proposed solely on the basis of the in vitro activities of plant glycosyl hydrolases. Received: 21 May 2000 / Accepted: 7 June 2000     

The genome of black raspberry (Rubus occidentalis)

Black raspberry (Rubus occidentalis) is an important specialty fruit crop in the US Pacific Northwest that can hybridize with the globally commercialized red raspberry (R. idaeus). Here we report a 243 Mb draft genome of black raspberry that will serve as a useful reference for the Rosaceae and Rubus fruit crops (raspberry, blackberry, and their hybrids). The black raspberry genome is largely collinear to the diploid woodland strawberry (Fragaria vesca) with a conserved karyotype and few notable structural rearrangements. Centromeric satellite repeats are widely dispersed across the black raspberry genome, in contrast to the tight association with the centromere observed in most plants. Among the 28 005 predicted protein‐coding genes, we identified 290 very recent small‐scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes which may be related to key agronomic traits during black raspberry domestication. This contrasts patterns of recent duplications in the wild woodland strawberry F. vesca, which show no patterns of enrichment, suggesting gene duplications contributed to domestication traits. Expression profiles from a fruit ripening series and roots exposed to Verticillium dahliae shed insight into fruit development and disease response, respectively. The resources presented here will expedite the development of improved black and red raspberry, blackberry and other Rubus cultivars.     

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.