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PubMed2002

Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis.

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZAT isolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5'-upstream sequences. Fusion of a 1,476-bp 5'-upstream sequence of PGAZAT to beta-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

Amino Acid Sequence↗
PubMed2002

Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion.

Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett [1990] Plant Cell 2: 1239-1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development.

Cell Adhesion↗
PubMed2003

Polygalacturonase beta-subunit antisense gene expression in tomato plants leads to a progressive enhanced wound response and necrosis in leaves and abscission of developing flowers.

Tomato (Lycopersicon esculentum var. Better Boy) plants were transformed with a tomato leaf wound-inducible polygalacturonase (PG) beta-subunit gene in the antisense orientation (PGbetaS-AS) under the control of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants exhibited small localized lesions, which eventually enlarged and spread throughout the entire surfaces of the leaves, resulting in cell death. The same lesions were also observed in the peduncle of developing flowers, extending to the whole flower causing abscission, resulting in a sterile phenotype. Leaves of transgenic plants exhibited elevated levels of PG activity, hydrogen peroxide, and enhanced defense signaling in response to wounding and elicitor treatment. The defense signaling increased was accompanied by an increased resistance toward tobacco hornworm (Manduca sexta) larvae. The cumulative results suggest that in the absence of the beta-subunit protein in tomato leaves, an increase in PG activity occurred that led to an enhanced wound response, the formation of lesions leading to severe necrosis, and an abscission of developing flowers.

Animals↗
PubMed2003

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation.

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.

Amino Acid Sequence↗
PubMed2005

The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.

Binding Sites↗
PubMed1997

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.

Amino Acid Sequence↗
PubMed1999

Expression of a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Gene Expression Regulation, Enzymologic↗
PubMed2000

Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma.

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes;Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to beta-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5'-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.

Ethylenes↗
PubMed1992

The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.

Amino Acid Sequence↗
PubMed1993

Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp.

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp.

Base Sequence↗
PubMed1994

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.

Amino Acid Sequence↗
PubMed2003

Resolving the space-group ambiguity of crystals of tomato fruit polygalacturonase.

The first polygalacturonase from a plant, tomato fruit PG2, has been crystallized and data have been collected to a resolution of 1.87 A. The autoindexing program strongly favors one of the primitive orthorhombic cells. A plausible molecular-replacement solution for two molecules in the asymmetric unit has been found for data assigned to space group P2(1)2(1)2(1). Although the numerical criteria and the electron-density maps are reasonable for this solution, manually adjusted models do not refine to an R factor below 0.48. Visual inspection of hkl Bragg planes does not reveal a breakdown in mm symmetry. Nevertheless, the correct space group has been determined to be P2(1), with similar unit-cell parameters, a beta angle of 90.04 degrees and four molecules in the asymmetric unit. The R(sym) of 0.053 for data processed in P2(1)2(1)2(1) is very similar to the R(sym) of 0.047 for the same data processed in P2(1). Comparisons of the intermediate results using the P2(1)2(1)2(1) and P2(1) data sets are provided and the subtle indications of an initial erroneous space-group assignment are discussed.

Crystallization↗
PubMed2000

Preliminary X-ray crystallographic analysis of a plant defence protein, the polygalacturonase-inhibiting protein from Phaseolus vulgaris.

A leucine-rich repeat plant protein involved in resistance to pathogens, a polygalacturonase-inhibiting protein (PGIP-1) from Phaseolus vulgaris, has been crystallized and preliminary X-ray characterization has been performed. The protein contains ten repeats of a short (24 amino-acid) leucine-rich repeat motif. Single crystals of the protein were grown from vapour-diffusion experiments using PEG 2K monomethylether as precipitant; these crystals diffract to at least 2.3 A resolution. The space group is P2(1), with two molecules of PGIP-1 in the asymmetric unit; the crystals contain approximately 38% solvent.

Amino Acid Sequence↗
PubMed1991

Characterization of a polygalacturonase gene of Aspergillus niger RH5344.

We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp coding for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C-terminal region probably comprising the elements of substrate binding and the catalytic centre.

Amino Acid Sequence↗
PubMed2005

Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing.

Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.

Alleles↗
PubMed2002

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris.

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.

Amino Acid Substitution↗
PubMed1990

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.

Aflatoxins↗
PubMed1995

Isolation and characterization of polygalacturonase genes (pecA and pecB) from Aspergillus flavus.

Two genes, pecA and pecB, encoding endopolyglacturonases were cloned from a highly aggressive strain of Aspergillus flavus. The pecA gene consisted of 1,228 bp encoding a protein of 363 amino acids with a predicted molecular mass of 37.6 kDa, interrupted by two introns of 58 and 81 bp in length. Accumulation of pecA mRNA in both pectin- or glucose-grown mycelia in the highly aggressive strain matched the activity profile of a pectinase previously identified as P2c. Transformants of a weakly aggressive strain containing a functional copy of the pecA gene produced P2c in vitro, confirming that pecA encodes P2c. The coding region of pecB was determined to be 1,217 bp in length interrupted by two introns of 65 and 54 bp in length. The predicted protein of 366 amino acids had an estimated molecular mass of 38 kDa. Transcripts of this gene accumulated in mycelia grown in medium containing pectin alone, never in mycelia grown in glucose-containing medium, for both highly and weakly aggressive strains. Thus, pecB encodes the activity previously identified as P1 or P3. pecA and pecB share a high degree of sequence identity with polygalacturonase genes from Aspergillus parasiticus and Aspergillus oryzae, further establishing the close relationships between members of the A. flavus group. Conservation of intron positions in these genes also indicates that they share a common ancestor with genes encoding endopolyglacturonases of Aspergillus niger.

Amino Acid Sequence↗