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PubMed1975

Polygalacturonase of Botrytis cinerea E-200 Pers.

A polygalacturonase (poly(1,4-alpha-D-galacturonide)glycanohydrolase, EC 3.2.1.15) was purified from the culture fluid of Botrytis cinerea. The polygalacturonase preparation, homogeneous on the basis of disc-gel electrophoresis also showed pectinesterase activity. Some properties of the purified polygalacturonase were studied. It had a molecular weight about 69 000. It was inactivated by p-chloromercuribenzoate, tetranitromethane and urea. A 50% loss in viscosity of sodium polypectate solution occurred when 4.6% of the glycosidic bonds were hydrolyzed. The only end product of sodium polypectate and oligogalacturonides hydrolysis was monogalacturonic acid.?

Chloromercuribenzoates↗
PubMed1980

Polygalacturonase Activity and Ethylene Synthesis during Cucumber Fruit Development and Maturation.

Polygalacturonase (EC 3.2.1.15) activity in seed cavity tissue from harvested cucumber fruit increased over 20-fold after the fruit had produced a transient burst of ethylene during maturation. This increase was observed in six cucumber cultivars and was present whether polygalacturonase activity was measured at pH 4.6 or 6.2. The seed cavity tissue pH decreased as polygalacturonase activity increased both in ripening fruit and in harvested immature fruit exposed to 10 microliters per liter ethylene in air.

Journal Article↗
PubMed1981

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings.

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis.

Journal Article↗
PubMed1981

Properties of Rhizopus stolonifer Polygalacturonase, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings.

Some properties of the polygalacturonase-elicitor from the filtrates of Rhizopus stolonifer cultures have been examined in an attempt to understand its mode of action as an elicitor of casbene synthetase activity in castor bean seedlings. Both the polygalacturonase activity and the elicitor activity are heat-labile with similar heat-sensitivity profiles. Also, the catalytic activity of the enzyme is lost on treatment with sodium periodate, as had been shown previously for the elicitor activity. The pH optimum of the enzyme activity with polygalacturonic acid as the substrate is 4.9. Exposures of germinating castor bean seedlings to the elicitor for short-term periods of 1 to 10 minutes followed by washing and incubation in sterile, distilled water are partially effective in elicitation in comparison with the continuous exposure of the seedlings over 11 hours to the same amount of the elicitor. The initial rate of reaction catalyzed by the enzyme is about 3 times faster with polygalacturonic acid as a substrate than with partially (50%) methylated polygalacturonic acid (pectin). The K(m) value of the enzyme for polygalacturonic acid is about 4.2 millimolar in terms of monomeric units and about 0.07 millimolar in terms of polymer concentration. Examination of the types of products formed by the action of the enzyme suggests that it is an endo-hydrolase. The amino acid composition of this enzyme is similar to those of other extracellular fungal proteins reported. The carbohydrate moiety of the glycoprotein polygalacturonase-elicitor is composed of 92% mannose and 8% glucosamine by gas chromatography-mass spectrometry analysis. The linkage group analysis of the carbohydrate moiety showed that mannosyl residues which are 1,2-linked comprise about 70% of the total glycosyl residues and demonstrated the presence of some 1,3,6- and 1,2,6-linked branching mannosyl residues.

Journal Article↗
PubMed1983

Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation.

Ultrastructural changes in the pericarp of tomato (Lycopersicon esculentum Mill) fruit were followed during ripening. Ethylene production was monitored by gas chromatography and samples analyzed at successive stages of the ripening process.Changes in the cytoplasmic ultrastructure were not consistent with the suggestion that ripening is a ;senescence' phenomenon. A large degree of ultrastructural organization, especially of the mitochondria, chromoplasts, and rough endoplasmic reticulum, was retained by ripe fruit.Striking changes in the structure of the cell wall were noted, beginning with dissolution of the middle lamella and eventual disruption of the primary cell wall. These changes were correlated with appearance of polygalacturonase (EC 3.2.1.15) isoenzymes. Application of purified tomato polygalacturonase isoenzymes to mature green fruit tissue duplicated the changes in the cell wall noted during normal ripening. Possible roles of the polygalacturonase isoenzymes in cell wall disorganization are discussed.

Journal Article↗
PubMed1990

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit.

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.

Journal Article↗
PubMed1991

Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress.

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O(2) regimes. Low O(2) concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O(2) did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O(2) but not in 7.5 or 10% O(2). The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O(2) tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O(2) levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O(2) levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O(2).

Journal Article↗
PubMed1991

Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3.

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.

Journal Article↗
PubMed1993

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen.

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.

Amino Acid Sequence↗
PubMed1994

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein.

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endo-PGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.

Amino Acid Sequence↗
PubMed1994

Expression of the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains capable of simultaneous secretion of bacterial glucanase and pectinase enzymes have been developed. The Butyrivibrio fibrrisolvens endo-beta-1,4-glucanase gene (end1), the Erwinia chrysanthemi pectate lyase gene (pelE) and E. carotovora polygalacturonase gene (peh1) were each inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into yeast-centromeric shuttle vectors. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of glucanase and pectinases was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). These YCplac111-based constructs, designated END1, PEL5, AND PEH1, respectively, were transformed into S. cerevisiae. The END1, PEL5 and PEH1 constructs were co-expressed in laboratory strains of S. cerevisiae as well as in wine and distillers' yeasts. DNA-RNA hybridization analysis showed the presence of END1, PEL5 and PEH1 transcripts. Carboxymethylcellulose and polypectate agarose assays revealed the production of biologically active endo-beta-1,4-glucanase, pectate lyase and polygalacturonase by the S. cerevisiae transformants. Interestingly, although the same expression-secretion cassette was used in all three constructs, time-course assays indicated that the pectinases were secreted before the glucanase. It is tempting to speculate that the bulkiness of the END1-encoded protein and the five alternating repeats of Pro-Asp-Pro-Thr(Gln)-Pro-Val-Asp within the glucanase moiety could be involved in the delayed secretion of the glucanase.

Amino Acid Sequence↗
PubMed1978

Polygalacturonase, biomass and ascospore production by Byssochlamys fulva. II. Effects of sugars found in fruits.

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with glucose, sucrose, or fructose was studied over a 20-day incubation period at 30 degrees C. The production of polygalacturonase was variable, but most activity was detected between 4 and 8 days in 1% sugar media at an initial pH of 4 or 5. The rate of biomass production was retarded early in the incubation period in media initially at pH 3 or 4 as compared to pH 5, but the amount of growth was about the same in media containing the test sugars after 20 days. Large numbers of ascospores were produced between 8 and 10 days in media containing 5% sugar initially at pH 5 and 4. Production of ascospores was retarded at pH 3 in media containing 5% sugar as compared to media initially at pH 5 and 4.

Ascomycota↗
PubMed2006

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

Acyltransferases↗
PubMed2003

Two Brassica napus polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses.

Plants encode a distinct set of polygalacturonase inhibitory proteins (PGIPs) that function to inhibit polygalacturonase enzymes produced by soft-rot fungal pathogens. We characterized two PGIP-encoding genes ( Bnpgip1 and Bnpgip2) from Brassica napus DH12075 (a double-haploid line derived from a cross between 'Crésor' and 'Westar'). The two proteins exhibit 67.4% identity at the amino acid level and contain 10 imperfect leucine-rich repeats. The pgip genes are present as a small multigene family in B. napus with at least four members. Bnpgip1 and Bnpgip2 are constitutively expressed in roots, stems, flower buds and open flowers. In mature leaf tissue, different levels of induction were observed in response to biotic and abiotic stresses. Bnpgip1 expression was highly responsive to flea beetle feeding and mechanical wounding, weakly responsive to Sclerotinia sclerotiorum infection and exposure to cold but not to dehydration. Conversely, Bnpgip2 expression was strongly induced by S. sclerotiorum infection and to a lesser degree by wounding but not by flea beetle feeding. Application of jasmonic acid to leaves induced both Bnpgip1 and Bnpgip2 gene expression; however, salicylic acid did not activate either gene. Taken together, these results suggest that separate pathways regulate Bnpgip1 and Bnpgip2, and that their roles in plant development or resistance to biotic and abiotic stress differ.

Amino Acid Sequence↗
PubMed2005

The active component in the flax-retting system of the zygomycete Rhizopus oryzae sb is a family 28 polygalacturonase.

The zygomycete Rhizopus oryzae sb is a very efficient organism for retting of flax, the initial microbiological step in the process of making linen. An extracellular polygalacturonase, when isolated could perform retting, and therefore probably is the key component in the retting system of R. oryzae. This was purified and characterized. The purified enzyme has a molecular mass of 37,436 Da from mass spectrometric determination, an isoelectric point of 8.4, and has non-methylated polygalacturonic acid as its preferred substrate. Peptide sequences indicate that the enzyme belongs to family 28, in similarity with other polygalacturonases (EC. 3.2.1.15). It contains, however an N-terminal sequence absent in other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium Ralstonia solanacearum. The biochemical background for the superior retting efficiency of R. oryzae sb is discussed.

Amino Acid Sequence↗
PubMed1990

Cloning and DNA sequence analysis of a polygalacturonase cDNA from Aspergillus niger RH5344.

A 1319 bp long cDNA encoding for a polygalacturonase (EC 3.2.1.15) from Aspergillus niger RH5344 comprises a single open reading frame of 1089 bp which includes the mature protein of 362 amino acids and an NH2-terminal signal peptide of 27 amino acids. The directly determined peptides of the mature polygalacturonase confirmed the sequence information deduced from the cDNA.

Amino Acid Sequence↗
PubMed1992

Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein.

The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp. carotovora has been cloned previously [Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044]. We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens. To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions. Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence. There was approximately fourfold variation in the production efficiency of B. subtilis strains carrying the different constructs. The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide. This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium. In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs. Our results suggest that fusions containing intact signal peptides, which are common when producing foreign proteins, are not necessarily the most efficient.

Amino Acid Sequence↗
PubMed2004

Purification and biochemical characterization of polygalacturonase II produced in semi-solid medium by a strain of Fusarium moniliforme.

A strain of Fusarium moniliforme isolated from a tropical mangrove ecosystem near Mumbai, India and deposited in the National Collection of Industrial Microorganisms (NCIM) as F. moniliforme NCIM 1276. The organism produced a single extracellular polygalacturonase (PG I) [EC 3.2.1.15] at pH 5 and a single pectate lyase (PL) [EC 4.2.2.2] at pH 8 in liquid medium containing 1% citrus pectin. Growth on semi-solid medium containing wheat bran and orange pulp resulted in a three-fold increase in PG production and a two-fold increase in PL production in comparison with that in liquid medium. The increased production of PG on semi-solid media, as compared to production in liquid media was investigated. The increased production of PG was partly due to the expression of a second polygalacturonase (PG II) isoenzyme by the fungus which was biochemically different from the one produced in liquid medium. The second PG II was a 30.6kDa enzyme, had an alkaline pI of 8.6, the Km was 0.166mg ml(-1), Vmax 13.33 micromol min(-1) mg(-1) and the kcat was 403 min(-1). It had a specific activity of 18.66U mg(-1). The differences between the PGs (PG I and PG II) suggest that the two enzymes are the products of different genes. The fungus also produced the same two PGs when it infected Lycopersicon esculentum (tomato). Only one PL was produced irrespective of growth conditions.

Culture Media↗