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At least 19 recordsLinked to original sources
PubMed1990

Analysis of tomato polygalacturonase expression in transgenic tobacco.

Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening. Polygalacturonase is synthesized as a glycoprotein precursor that undergoes numerous cotranslational and post-translational processing steps during its maturation, yielding three isozymes in tomato fruit, PG1, PG2A, and PG2B. To investigate the physiological roles of the three isozymes and the functional significance of the polygalacturonase processing domains in its intracellular transport and activity, we have examined polygalacturonase expression in transgenic tobacco plants. A full-length polygalacturonase cDNA was placed under control of the cauliflower mosaic virus 35S promoter and introduced into tobacco by way of Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants indicated that (1) immunologically detectable polygalacturonase can be extracted from leaves, roots, and stems of transgenic tobacco plants; (2) only PG2A and PG2B were detectable in transgenic tobacco; (3) the polygalacturonase isozymes present in transgenic tobacco were electrophoretically indistinguishable from the tomato isozymes; (4) the N-terminal sequence, degree of N-linked glycosylation, and extent of oligosaccharide processing were similar in polygalacturonase from transgenic tobacco and tomato; (5) polygalacturonase was properly localized in cell walls of transgenic tissue; (6) the protein was enzymatically active in vitro; however, (7) accumulation of PG2A and PG2B in cell walls of transgenic tobacco did not result in pectin degradation in vivo. These results indicated that tomato polygalacturonase was properly processed and transported to the cell wall of tobacco. However, accumulation of the two polygalacturonase isozymes expressed in this heterologous host was insufficient to promote polyuronide degradation in tobacco leaf tissue.

Amino Acid Sequence↗
PubMed1989

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Base Sequence↗
PubMed1992

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.

Amino Acid Sequence↗
PubMed1991

Polygalacturonase is a virulence factor in Agrobacterium tumefaciens biovar 3.

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grapes. All biovar 3 strains, regardless of their tumorigenicity, produce in culture a single polygalacturonase with a pI around 4.5. A. tumefaciens biovar 3 strain CG49 was mutagenized with Tn5 by using pSUP2021 as a suicide vector. A mutant strain, CG50, lacking polygalacturonase activity was isolated. The mutation was due to a single Tn5 insertion in an 8.5-kb EcoRI fragment that also contained the polygalacturonase structural gene. The polygalacturonase-encoding pehA gene was cloned in Escherichia coli by using the plasmid pBluescript as a vector. Activity-stained isoelectric focusing gel analysis demonstrated that E. coli cells harboring the pehA+ recombinant plasmid pCPP2067 produced a polygalacturonase in culture with the same pI as the enzyme produced by CG49. The pehA gene was localized within a 2.5-kb HindIII-SalI fragment. This fragment was used as a probe in Southern hybridization analysis and showed that no closely related genes are present in A. tumefaciens biovars 1 or 2, Rhizobium leguminosarum, or Bradyrhizobium japonicum. The polygalacturonase mutant was unable to induce root decay in grapes (Vitis vinifera cv. Chardonnay) and was substantially less tumorigenic than the wild type in grape stems when low levels of inoculum were used, although both strains were equally tumorigenic in potato disc assays. The results indicate that polygalacturonase is a virulence factor in both the root decay and crown gall incited in grapes by A. tumefaciens biovar 3.

Agrobacterium tumefaciens↗
PubMed1990

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger.

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.

Alginates↗
PubMed1991

Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity.

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].

Chromatography↗
PubMed1978

[Mode of action and inhibition of polygalacturonase covalently bound to polysaccharide and glass carriers].

Endo-polygalacturonase (EC 3.2.1.15.) from Aspergillus spec. is much changed as far as its mode of action and the interaction with vegetable inhibitors of pectinase (from green beans and cucumbers) are concerned when it is covalently bound to insoluble carriers (Sepharose, cellulose powder, macroporous glass and nonporous ballotinis). Whereas a 2% degradation of substrate by the soluble enzyme caused a 50% decrease of viscosity of citrus pectic acid, the comparable degradation of substrate was increased to a level of about 10% with the investigated polygalacturonase carrier complexes apparently independent of the properties of the carriers and the kind of binding of the enzyme. In contrast to this the higher degradation of substrate of 15 and 20% respectively which was further stated at a 50% decrease of viscosity is unambiguously connected with the carriers and is in direct correlation with the specific activity of the polygalacturonase carrier complexes. Contrary to the soluble enzyme the covalently bound enzyme produces more lower oligomerous galacturonic acids by an exo-mechanism or by multiple attack already at the beginning of the hydrolysis of pectic acid. During the final stage there is an enrichment of trigalacturonic acid besides mono- and digalacturonic acids independent of the state of solution of the enzyme. It could further be stated that the strong inhibition of the soluble endo-polygalacturonase by selected pectinase inhibitors which was described earlier is reduced by degrees with the enzyme covalently bound to the insoluble carriers.

Enzymes, Immobilized↗
PubMed1992

Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production.

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

Cloning, Molecular↗
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↗
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↗
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↗
PubMed1990

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.

Amino Acid Sequence↗
PubMed1988

Analysis of the Pseudomonas solanacearum polygalacturonase encoded by pglA and its involvement in phytopathogenicity.

A major endopolygalacturonase excreted by Pseudomonas solanacearum was purified to greater than 95% homogeneity and shown to have an isoelectric point of 9.0 and a subunit molecular mass of 52 kilodaltons (kDa). The gene encoding this enzyme (pglA) was isolated from a genomic library of P. solanacearum DNA based on its expression in Escherichia coli and shown to be contained on a 1.8-kilobase DNA fragment. The identity of the pglA gene product and the 52-kDa polygalacturonase was demonstrated by immunoadsorption and isoelectric focusing experiments. The cloned pglA gene was apparently expressed from its own promoter in E. coli and its product was partially secreted into the periplasm. The pglA gene was insertionally inactivated in vitro and used to mutate the chromosomal pglA gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant strain was deficient in production of the 52-kDa polygalacturonase and took twice as long to wilt and kill tomato plants as the wild-type parent in plant bioassay experiments. Complementation in trans with the wild-type cloned pglA gene restored virulence to near wild-type levels. The data indicate that the pglA gene is important, but not absolutely necessary, for pathogenesis.

Escherichia coli↗
PubMed1990

[Production of polygalacturonase by Bacillus subtilis cultured with waste and residues as carbon sources].

In the recent study, it was observed that if when the initial pH was 7.0, incubation temperature was 37 degrees C and aeration (150 rpm) was supplied polygalacturonase production by Bacillus subtilis reached to its maximum. Molasses, Vinase and Peach or Apricot pomace were used as carbon sources at differing concentrations and the highest differential rate of enzyme synthesis was detected in the medium prepared with Vinase having a 5% of total sugar content (6.74 delta U/delta O.D.). It also was detected that the waste or residues mentioned above could be used as carbon sources for the polygalacturonase enzyme production by Bacillus subtilis.

Bacillus subtilis↗
PubMed1990

DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.

The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.

Amino Acid Sequence↗
PubMed1992

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach.

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene. Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA form in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

Blotting, Northern↗
PubMed1991

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize.

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Amino Acid Sequence↗