Antisense suppression of tomato endo-1,4-β-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4--glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.     

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.     

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.     

Expression of fungal thermotolerant endo-1,4-β-glucanase in transgenic barley seeds during germination

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4--glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI -amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. A decrease in the soluble -glucan content in the wort improves the filtration rate of beer.     

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.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

Growth enhancement of transgenic poplar plants by overexpression of Arabidopsis thaliana endo-1,4–β-glucanase (cel1)

Poplar (Populus tremula) plants which had been transformed with Arabidopsis thaliana cel1 cDNA and successfully over-expressed the gene, exhibited significant phenotypic alterations which included taller plants, larger leaves, increased stem diameter, wood volume index, dry weight and a higher percentage of cellulose and hemicellulose, compared to the wild-type plants. Transgenic A. thaliana plants over-expressing A. thaliana cel1 exhibited similar levels of cel1 mRNA in the elongation zone of the flowering stem and higher levels in mature leaves when compared with wild-type plants. CEL1 protein levels in the elongation zone of the flowering stem of transgenic plants were similar or slightly higher compared to that of the wild-type plants, whereas mature leaves of transgenic plants contained a higher level of CEL1. These data indicate that in elongating zone of Arabidopsis, CEL1 level is tightly regulated. In contrast to transgenic poplar over-expressing the A. thaliana cel1, no phenotypic difference was found between A. thaliana transgenic and wild-type plants.     

Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein⁻¹, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.     

Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k _cat/K _m?=?3,590 mg mL^?1 s^?1) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K _m and V _max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min⁻¹ mg⁻¹, respectively. These characteristics indicate that Cel5G has potential for industrial use.     

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-β-glucanase

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.     

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase

β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.     

LeMAN4 endo-β-mannanase from ripe tomato fruit can act as a mannan transglycosylase or hydrolase

Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.