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PubMed2006

Role of poly-galacturonase inhibiting protein in plant defense.

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to play an important role in the defense against plant pathogen fungals. PGIPs are glycoproteins located in plant cell wall which reduce the hydrolytic activity of polygalacturonases (PGs), limit the growth of plant pathogens, and also elicit defense responses in plant. Furthermore, PGIPs belong to the super family of leucine reach repeat (LRR) proteins which also include the products of several plant resistance genes. Many of the studies show the PGIP properties, molecular characteristics, and PGIP gene expression induced by some elicitors. Some of the studies review individual PGIP gene expression in different signal transduction pathways. This article summarizes the properties, different signal transduction mechanisms, detecting methods, transgenic plants, and function of PGIP. It also presents PGIP gene expression in different stages of maturity, tissues, and varieties. The review especially reports the particular PGIP gene expression induced by different biotic and abiotic stresses, offers some questions, and prospects the future study, which are needed in order to develop efficient strategies for disease-resistant plants. They may be useful for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection, which decrease the use of insecticide.

Plant Physiological Phenomena↗
PubMed1994

Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and nonmelting peach cultivars.

Two distinct partial cDNAs, PRF1 and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica L. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRF5, from a lambda ZAP II cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRF5 included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20 degrees C and coincided with the climacteric peak in ethylene evolution. Levels of 1-aminocyclopropane-1-carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.

Amino Acid Sequence↗
PubMed1989

Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.

Aspergillus nidulans↗
PubMed1989

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere.

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.

Genetic Engineering↗
PubMed1990

Common amino acid domain among endopolygalacturonases of ascomycete fungi.

The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved.

Amino Acid Sequence↗
PubMed1992

Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii.

The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845, consistent with previous results of Morales et al. (V. Morales, E. Martínez-Molina, and D. Hubbell, Plant Soil 80:407-415, 1984). However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym- ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843. Isoelectric focusing activity gels revealed one ANU843 polygalacturonase isozyme with a pI of approximately 7.2. These studies show that R. leguminosarum bv. trifolii produces multiple enzymes that cleave glycosidic bonds in plant cell walls and that are cell bound.

Carboxymethylcellulose Sodium↗
PubMed2005

[Changes in cell wall component metabolism and ultrastructure of postharvest persimmon fruit during softening].

The changes in cellular wall hydrolases, cellular wall components and cellular wall ultrastructure of postharvest persimmon (Diospyros kaki L. cv. Bianhua) fruit during softening were studied. Pectinesterase activity increased sharply at first and reached a peak (Fig.3A). Significant correlation was observed between the pectinesterase activity and the loss of flesh firmness (r= -0.74). Polygalacturonase activity increased slowly (Fig.3B), but there was no significant correlation between the polygalacturonase activity and the loss of flesh firmness. Beta-galactosidase activity increased sharply (Fig.3C), with a negative correlation between the b-galactosidase activity and the loss of flesh fruit firmness (r= -0.77). Cellulase activity increased markedly during ripening (Fig.3D). Significant correlation was observed between cellulase activity and the loss of flesh firmness (r= -0.90). Consistent with the increases in activity of cell wall hydrolases of cell wall constituents, fruit softening was accompanied by a progressive increase in WSP (water soluble pectin) content and a progressive decrease in protopectin and cellulose content (Fig.4). The cell wall structure was integrated when persimmon was harvested (Fig.5A). After 3 d of ripening, the middle lamella became liquefied (Fig.5B), or even the primary cell wall was dissolved in some regions (Fig.5C).

Cell Wall↗
PubMed1991

Using antisense RNA to study gene function.

Over the past two years, antisense RNA technology has been developed in plants in several experimental systems. Progress reported in the literature will be reviewed. Special emphasis will be placed on experiments in which antisense RNA has been used to generate novel tomato mutants through the downregulation of polygalacturonase (PG), a major cell wall hydrolase. This work has shown that antisense RNA inhibits PG expression specifically without affecting the expression of other genes. The antisense gene and its phenotypic expression are stably inherited. The inhibitory effect of an antisense gene can be enhanced by increasing the copy number of the gene. The interaction of the antisense gene with its target is only seen when the target gene is expressed. Antisense RNA technology has been used to clarify the biochemical function of polygalacturonase and its role in tomato fruit softening. Recently, this approach has also been applied to elucidate the biochemical role of another gene from tomato, pTOM13, whose function was previously unknown. Thus, antisense RNA technology can now be used to generate novel plant mutants that will make possible the definition of the biochemical and biological role of genes whose functions are otherwise unknown.

Genes, Plant↗
PubMedDate not supplied

[Ecologic features of cultures of the species Clostridium gürfelii].

The Clostridium cultures decomposing pectin were isolated from flax straw during flax retting. The activity was manifested only at high pH values, the optimum pH being 6.7--8.2. Such ecological adaptation of the cultures is determined by the properties of the pectolytic enzyme complex which they synthesize. The complex does not contained polygalacturonase (the optimum pH 5.0) but has a high activity of pectate transeliminase (the optimum pH 8.0--8.4). The cultures differ from Clostridium felsineum which possesses a high activity of polygalacturonase and therefore can decompose pectin in acid medium. By their morphological, cultural and physiological properties, the bacteria are classed as Clostridium güfelii (Prévot, 1966).

Carboxylic Ester Hydrolases↗
PubMed1998

Regulation of pectic enzymes from the exo-1 mutant strain of Neurospora crassa: effects of glucose, galactose, and galacturonic acid.

The exo-1 mutant of Neurospora crassa produced and secreted pectolytic activities when incubated in the presence of pectin-containing biological materials. This study shows that polygalacturonase, pectate lyase and pectin lyase activities were induced in media supplemented with galactose or galacturonic acid, indicating that these sugars induced the synthesis of pectinases. Pectinesterase activity was undetectable. Polygalacturonase activity was better induced by galactose than by galacturonic acid. The reverse was true for lyase activities. The inducing effect of galactose and galacturonic acid seemed to be different: (i) a mixture of galactose and galacturonic acid synergistically increased the production of pectic enzymes, as compared to that in the presence of one of these sugars; (ii) the inducing effect of galacturonic acid was partially repressed by glucose; (iii) in contrast, the inducing effect of galactose, rather than repressed, was enhanced by the presence of glucose. Altogether, these data point out to a complex mechanism of regulation of pectolytic enzymes by pectin-containing organic substances.

Enzyme Induction↗
PubMed2000

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues.

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.

Culture Media↗
PubMed2006

Characterization of expressed Pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat.

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. A number of PGIPs have been characterized from dicot species, whereas only a few data are available from monocots. Database searches and genome-specific cloning strategies allowed the identification of four rice (Oryza sativa L.) and two wheat (Triticum aestivum L.) Pgip genes. The rice Pgip genes (Ospgip1, Ospgip2, Ospgip3 and Ospgip4) are distributed over a 30 kbp region of the short arm of chromosome 5, whereas the wheat Pgip genes, Tapgip1 and Tapgip2, are localized on the short arm of chromosome 7B and 7D, respectively. Deduced amino acid sequences show the typical LRR modular organization and a conserved distribution of the eight cysteines at the N- and C-terminal regions. Sequence comparison suggests that monocot and dicot PGIPs form two separate clusters sharing about 40% identity and shows that this value is close to the extent of variability observed within each cluster. Gene-specific RT-PCR and biochemical analyses demonstrate that both Ospgips and Tapgips are expressed in the whole plant or in a tissue-specific manner, and that OsPGIP1, lacking an entire LRR repeat, is an active inhibitor of fungal polygalacturonases. This last finding can contribute to define the molecular features of PG-PGIP interactions and highlights that the genetic events that can generate variability at the Pgip locus are not only limited to substitutions or small insertions/deletions, as so far reported, but can also involve variation in the number of LRRs.

Amino Acid Sequence↗
PubMed2003

Transformation of apple ( Malus domestica Borkh.) with the stilbene synthase gene from grapevine ( Vitis vinifera L.) and a PGIP gene from kiwi ( Actinidia deliciosa).

The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs. Elstar and Holsteiner Cox in order to establish resistance against fungal diseases. The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi ( Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation. A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis. High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.

Acclimatization↗
PubMed1988

Structure and anti-complementary activity of pectic polysaccharides isolated from the root of Angelica acutiloba Kitagawa.

Four pectic polysaccharides (AR-2IIa-IId) with anti-complementary activity have been isolated from a hot-water extract of the root of Angelica acutiloba Kitagawa. Each of these polysaccharides contained a large proportion of GalA together with neutral sugars consisting mainly of Rha, Ara, and Gal. Digestion with endo-alpha-(1----4)-polygalacturonase indicated that AR-2IIa-IIc each contained a large proportion of enzyme-sensitive polygalacturonan regions, and that AR-2IId contained a large proportion of enzyme-resistant regions. When AR-2IId was de-esterified, it became sensitive to the enzyme. These polysaccharides also contained small proportions of enzyme-resistant regions (PG-1) which were rich in neutral sugars. Methylation analysis and base-catalysed beta-elimination studies suggested that each PG-1 contained a rhamnogalacturonan moiety in which 2,4-disubstituted Rha was attached to 4-substituted GalA through position 2 of Rha. Carboxyl-reduction and methyl- and de-esterification of these polysaccharides modulated their anti-complementary activities. Digestion with endo-alpha-(1----4)-polygalacturonase decreased the activities of AR-2IIa and -2IIb, but not those of AR-2IIc and -2IId. Although PG-1 fractions from AR-2IIa-IIc were more active than the original polysaccharides, oligogalacturonide fragments obtained by enzymic digestion had weak or negligible activity. AR-2IIa-IIc expressed their anti-complementary activities mainly via the classical pathway, but AR-2IId and each PG-1 expressed their activities via both the classical and alternative pathways.

Complement Activation↗
PubMed1991

Pollen specific cDNA clones from Zea mays.

We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-ripening polygalacturonase and to a pollen-specific polygalacturonase from Oenothera. The other three clones have no significant homologies to any reported sequence.

Amino Acid Sequence↗
PubMed1993

The pectic polysaccharide from Bupleurum falcatum L. enhances immune-complexes binding to peritoneal macrophages through Fc receptor expression.

Binding of glucose oxidase-anti-glucose oxidase complexes (GAG), a model of immune complexes, to macrophages was enhanced by treatment with an acidic pectic polysaccharide, bupleuran 2IIb, from Bupleurum falcatum L. GAG binding to macrophages by bupleuran 2IIb increased in a dose-dependent fashion, and was abolished when the Pronase-treated macrophages were incubated with bupleuran 2IIb. The GAG binding enhancing activity of bupleuran 2IIb was reduced by periodate oxidation but not Pronase digestion of bupleuran 2IIb. When bupleuran 2IIb was digested with endo-polygalacturonase, the resulting enzyme resistant carbohydrate portion showed potent activity. Scatchard analysis indicated enhanced expression of the Fc receptor (FcR) on the surface by the action of bupleuran 2IIb. The enhancement of GAG binding by bupleuran 2IIb was inhibited by the presence of actinomycin D or cycloheximide. Bupleuran-2IIb-stimulated cells showed enhanced expression of both FcRI and FcRII mRNA, which were measured as PCR products. These results suggested that the endo-polygalacturonase resistant carbohydrate portion of bupleuran 2IIb is important for the expression of the activity, and that the activity of bupleuran 2IIb on GAG binding was mediated by receptors for polysaccharide on the cells. The up-regulation of the Fc receptor by bupleuran 2IIb was also suggested to mediate by de novo synthesis of the receptor protein.

Animals↗
PubMed1999

Aspergillus kawachii produces an acidic pectin releasing enzyme activity.

A pectin-releasing (protopectinase, PPase) activity was found in a culture filtrate of Aspergillus kawachii IFO 4308. PPase activity was highest in the pH range of 2.0-2.5 and it was highly stable at 50 degrees C (85% of residual activity was found after a 10-h incubation in citrate-phosphate buffer, pH 3.0). Among other different enzyme activities, which are usually involved in plant cell-wall degradation, only polygalacturonase activity was detected. This result suggests that the PPase activity could correspond to a particular kind of polygalacturonase. Pectin extraction from lemon peels carried out at 50 degrees C for 2 h (pH 3.5) gave yields of ethanol-precipitated pectin equivalent to 17.4% of the initial total solids contained in the peels. Thus, this enzyme activity would allow carrying out a pectin extraction process at lower reaction pHs and higher temperatures in comparison with similar reports using other PPases. These properties seem to be very interesting from the practical point of view.

Journal Article↗
PubMed2000

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Allergens↗