Constitutive overexpression of a ripening-related pepper endo-1,4-β-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening

The ripening-related pepper endo-1,4--D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

Effect of simultaneous down-regulation of pectate lyase and endo-β-1,4-glucanase genes on strawberry fruit softening

Strawberry is a soft fruit with a short postharvest shelf-life. The loss of fruit firmness during ripening is mainly due to the disassembly of parenchyma cell walls mediated by the expression of genes encoding enzymes acting on pectins, such as pectate lyase, or hemicellulose, e.g. endo-β-1,4-glucanase. To determine if the simultaneous down-regulation of FaplC and FaEG3 genes, encoding a pectate lyase and a endo-β-1,4-glucanase, respectively, exerted an additive effect on strawberry softening, transgenic plants expressing tandem antisense sequences of both genes under the control of the constitutive promoter CaMV35S were generated. Fifteen independent transgenic lines were obtained and fruit yields and several quality parameters of transgenic ripe fruit were recorded during two consecutive years. Fruit yield was reduced in most of the lines, especially in the first evaluation period, and five out of 15 lines (33 %) did not set fruit. The expression of FaplC and FaEG3 genes was measured in ripe fruits from six selected lines showing the highest fruit yields. All selected lines showed a high level of FaplC gene silencing, ranging from 97 to 71 %; however, FaEG3 gene expression was only significantly down-regulated in two lines. Fruit colour and soluble solids contents were similar in control and transgenic ripe fruits, while fruit weight was slightly lower than control in some of the lines. In all lines, transgenic fruits were significantly firmer than control, with an increase in firmness ranging from 19 to 32 %. The reduction of fruit softening in transgenic fruits was not correlated with the suppression of FaEG3 gene expression, and lines with the highest simultaneous down-regulation of FaplC and FaEG3 showed similar fruit firmness to lines where only FaplC was suppressed. These results indicate that pectate lyase and endo-β-1,4-glucanase do not act in an additive or synergistic way during strawberry softening, and question the role of glucanases in this process.     

Mannans in tomato fruit are not depolymerized during ripening despite the presence of endo-β-mannanase

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.     

Absence of endo-1,4-β-glucanase KOR1 alters the Jasmonate-dependent defence response to Pseudomonas syringae in Arabidopsis

During plant–pathogen interactions, the plant cell wall forms part of active defence against invaders. In recent years, cell wall-editing enzymes, associated with growth and development, have been related to plant susceptibility or resistance. Our previous work identified a role for several tomato and Arabidopsis endo-1,4-β-glucanases (EGs) in plant–pathogen interactions. Here we studied the response of the Arabidopsis thaliana T-DNA insertion mutant lacking EG Korrigan1 (KOR1) infected with Pseudomonas syringae. KOR1 is predicted to be an EG which is thought to participate in cellulose biosynthesis. We found that kor1-1 plants were more susceptible to P. syringae, and displayed severe disease symptoms and enhanced bacterial growth if compared to Wassilewskija (Ws) wild-type plants. Hormonal and gene expression analyses revealed that the jasmonic acid (JA) pathway was activated more in kor1-1 plants with an increase in the JA-biosynthesis gene LOX3 and a greater accumulation of JA. Upon infection the accumulation of JA and JA-isoleucine (JA-Ile) was higher than in wild-type plants and increased the induction of LOX3 and the JA-responsive PDF1.2 gene. In addition, the increase of salicylic acid (SA) in healthy and infected kor1-1 may reflect the complex interaction between JA and SA, which results in the more susceptible phenotype displayed by the infected mutant plants. Callose deposition was enhanced in infected kor1-1 and an increase in pathogen-induced hydrogen peroxide took place. The susceptible phenotype displayed by KOR1-deficient plants was coronatine-independent. No significant changes were detected in the hormonal profile of the kor1-1 plants infected by coronatine-deficient P. syringae cmaA, which supports that absence of EG KOR1 alters per se the plant response to infection. We previously reported increased resistance of kor1-1 to B. cinerea, hence, the lack of this EG alters cell wall properties and plant responses in such a way that benefits P. syringae colonisation but restricts B. cinerea invasion.     

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo--mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo--mannanase in the endosperm after germination is induced by GAs, but the expression of endo--mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo--mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo--mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.Abbreviations ABA cis/trans-abscisic acid - GA(s) gibberellin(s) - IEF isoelectric focussing - pI(s) isoelectric point(s) We thank Dr. Bruce Downie for the seemingly endless but inspiring discussions.     

Developing tomato seeds when removed from the fruit produce multiple forms of germinative and post-germinative endo-β-mannanase. Responses to desiccation, abscisic acid and osmoticum

Several isoforms of endo-1,4-D-mannanase (EC3.2.1.78) are produced in the endosperm and embryo of tomato (Lycopersicon esculentum Mill.) seed prior to the completion of germination. Other isoforms appear in the embryo and in the lateral endosperm following germination. This occurs in seeds removed from the fruit prior to completion of development at 45 d after pollination and placed directly on water, or following drying. Hence desiccation is not required to induce either germination- or post-germination-related mannanase activity. Incubating seeds in abscisic acid or osmoticum results in a reduction of both germination and total mannanase activity, but the isoforms that are produced in the embryo and micropylar region of the endosperm are identical to those produced in water-imbibed seeds prior to germination. Incubation of seeds in a high concentration of abscisic acid prevents all enzyme production. Only after the completion of germination does mannanase increase in the lateral regions of the endosperm. In contrast, mannanase is produced in the micropylar region regardless of whether the seed germinates or not. The isoforms produced in the two regions of the endosperm are different, those in the lateral endosperm being more similar to those produced in the cotyledons and axes of the embryo. Embryos and endosperms dissected prior to completion of germination and incubated separately produce far fewer isoforms than when these parts are together in the intact seed.Abbreviations ABA cis-abscisic acid - DAP days after pollination - GA gibberellin - IEF isoelectric focusing - PEG polyethyleneglycol - pI isoelectric point This work was supported by Natural Sciences and Engineering Council of Canada grant A2210. B.V. received a fellowship from the Deutscher Akademischer Austauschdienst for her research at the University of Guelph. We are grateful to Dr. H.W.M. Hilhorst, Wageningen, for his critical comments.     

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> maximizes enzyme activity and irreversibly binds to amorphous cellulose

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.