Role Of Polygalacturonase Research Articles

Functional analysis of polygalacturonase gene RsPG2 from Rhizoctonia solani, the pathogen of rice sheath blight

Rice sheath blight, which is caused by Rhizoctonia solani, is a devastating disease in many areas of the world where rice is cultivated. The role of polygalacturonase (PG) in the infection process of R. solani was investigated in the present study by cloning and investigating the function of a new PG-encoding gene, which was designated RsPG2. Bioinformatics showed that RsPG2 consisted of a 1,633 bp open reading frame (ORF) with six introns; the deduced protein contained 436 amino acids with a predicted mass of 45.88 kDa after cleavage of the predicted 17-amino acid signal sequence. Culture supernatants obtained from Pichia pastoris producing recombinant RsPG2 degraded polygalacturonic acid in vitro, thus confirming that RsPG2 encodes a PG gene. Purified recombinant RsPG2 also degraded rice tissue and elicited obvious necrotic symptoms 48 h after inoculation. Real-time qRT-PCR indicated that the expression of RsPG2 was strongly induced during the infection of rice by R. solani. These data unequivocally demonstrate that RsPG2 plays an important role in R. solani infection and provide further insights for elucidating the function of pg genes in the virulence of R. solani.

Comparative analysis of polygalacturonase in the fruit of strawberry cultivars.

The role of polygalacturonase (PG) in the development, ripening, and softening of fruit from two strawberry cultivars with different flesh firmness and softening characteristics was compared. Changes in PG activity and gene expression during development, ripening and softening were measured. The PG genes from each cultivar were cloned and analyzed, and were classified with other PG genes using phylogenetic analysis. In Toyonoka fruit, PG activity increased gradually, reaching a peak during the pink stage, and remained at this level during post-harvest softening. Changes in PG gene expression were consistent with PG activity in these softer fruits. In the firmer Sweet Charlie fruits, PG activity was detected during the initial development stage, reaching a peak at the white stage, thereafter decreasing gradually with ripening and remaining at this lower level throughout softening. Changes in PG gene expression and PG activity were not consistent in these fruit. For both Toyonoka and Sweet Charlie PG genes (FaTPG and FaSCPG, respectively), the open reading frame was 1218 bp, encoding 405 amino acids. Five different nucleotide sites were observed between the two sequences, leading to two amino acid sequence mutations. FaTPG, FaSCPG, and PG genes from the Fragaria vesca genome were classified into three clades using phylogenetic analysis. The clade containing PG genes involved in fruit softening had functional similarity but there were no functional differences between FaTPG and FaSCPG. Differences in PG activity, gene sequence, and gene expression may have led to different roles of PG during ripening and softening in strawberries with different textures.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5–6, with an optimum at pH 4.5. PG activity was detectable 0–70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca2+, Mg2+, Mn2+ and Fe2+ and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.

Ethylene-sensitive and ethylene-insensitive abscission in Dendrobium: Correlation with polygalacturonase activity

We previously showed that the abscission of floral buds of the orchid Dendrobium cv. Miss Teen was absent in controls but highly promoted by ethylene, whilst open flowers also did not abscise without ethylene but were not responsive to ethylene. Here, we determined the activities of three enzymes in the abscission zone (AZ): polygalacturonase (PG), β-1,4-glucanase (cellulase), and pectin methylesterase (PME). In the AZ of floral buds an increase in PG activity coincided with the ethylene-induced abscission. A small increase was found in the AZ β-1,4-glucanase (cellulase) activity, shortly after the end of ethylene treatment of the flower buds and a few days before the onset of abscission. The activity of pectin methylesterase (PME) in the AZ of floral buds was not affected by ethylene. In the AZ of flowers that had just opened no increase in enzyme activity was observed after ethylene treatment. The data indicate a role of PG, and possibly of β-1,4-glucanase, in abscission. The data also suggest that the reason for the decrease in ethylene sensitivity of the abscission zone, during flower opening, lies upstream from the induction of enzyme activity in the AZ.

Changes in cell wall polysaccharides and cell wall degrading enzymes during ripening of Fragaria chiloensis and Fragaria ×ananassa fruits

Chilean strawberry ( Fragaria chiloensis ) is arising as a new fruit crop that can diversify the world supply of berries. Fruit softening is thought to be linked to an extensive modification of pectin and hemicellulosic cell wall fractions which in turn is determined to a great extent by the action of some cell wall-modifying enzymes. The objective of this work was to compare cell wall changes and the enzyme activities of pectin methylesterase (PME), polygalacturonase (PG), endoglucanase (EGase), α-arabinofuranosidase (AFase), β-galactosidase (βGal), and β-xylosidase (βXyl) between F. chiloensis and Fragaria × ananassa (cv. Chandler) at three fruit developmental stages: large green (LG), turning (T) and ripe (R). A rapid decrease in fruit firmness between LG and T stages was observed in both species; nevertheless firmness reduction in F. chiloensis was steeper than in F . × ananassa . This could be related to a faster loss of HCl-soluble polymers (HSP fraction) presented in F. chiloensis fruit. However, commercial F . × ananassa showed a greater depolymerization of HSP polymers than F. chiloensis . With the exception of PME, the activity of all assayed enzymes was higher in F. chiloensis than in F . × ananassa fruits. The role of PG and EGase seems to be central in both species: these enzymes are correlated to the decrease in the content of HSP and NaOH-soluble polymers (NSP fraction), respectively. In addition, activity levels of βGal and βXyl were significantly correlated with fruit firmness reduction in F. chiloensis and F. × ananassa , respectively.

Relationship of colour and other quality parameters of sweet cherry during development and ripening

Sweet cherry fruits ( Prunus avium cv. Vera, Carmen, Linda and Krupnoplodnaja) were harvested in different ripeness stages after anthesis 39, 42, 49, 56 days (Carmen); 37, 40, 47, 52, 59 days (Krupnoplodnaja); 32, 36, 44, 53, 58 days (Linda); 38, 42, 50, 52, 57 days (Vera). The colour, total soluble solids, dry matter content, β-galactosidase and polygalacturonase activity were determined. The dry matter content and total soluble solids content (Brix o ) increased during development. The L* values significantly decreased as a function of ripening while a* values increased up to 3 rd harvest, then they decreased, b* values continuously decreased as a function of development. The a*/b* values linearly increased as a function of development, indicating reddening of fruits. Hue angle (h o ) increased rapidly between harvest one and two. The measured (a*, b*, L*) and calculated parameters (h o , a*/b*, chroma) well represented the colour development of sweet cherries. Linear and strong correlation was found between a*/b*, L* values and the Brix o , exponential correlation was found between h o , chroma and Brix o . Activity of β-galactosidase was different among cultivars and as a function of development. The two maximums might β-galactosidase isoenzymes. The role of PG (polygalacturonase) could not be explained clearly enough, further investigations are needed to find the exact role of the enzyme in the cell wall metabolism of sweet cherry.

Host- and symptom-specific pectinase isozymes produced by Botrytis cinerea

Apple fruits and grapevine leaves were inoculated with four wild-type strains of Botrytis cinerea. Two types of symptoms were observed on apple, firm or soft rot. Extracts from infected tissues were analysed for polygalacturonase (PG) and pectin methylesterase (PME) isozyme profiles. They showed that different specific PG isozymes occur in grapevine leaves and in soft rot on apple fruit. PG and PME profiles were also studied in genetic transformants altered in their colonization ability and/or the type of rot that they caused on apple fruit. No PG activity was found in apples infected by firm-rotting wild-type strains or transformants, whereas two neutral PG isozymes (pls 7.3 and 7.6) were expressed in soft-rotting strains. No association was found between the production of these PG isozymes and the colonization level. The PME patterns were not correlated with the rotting type nor the colonization level. In vitro , firm-rotting strains and transformants showed no PG activity in glucose medium whereas soft-rotting ones produced several PG isozymes. In polygalacturonic acid cultures, all strains and transformants produced a single, common acidic isozyme. Unlike PG, no variation in the PME isozyme patterns in vitro could be correlated with the culture substrate on apple or the rotting type. The variation in pectinase profiles between in vitro and in planta contexts and the role of PGs in B. cinerea pathogenicity on apple fruit is discussed. We suggest that neutral PG isozymes are involved in the soft-rotting type on apple fruit.

Inhibition of respiration, ethylene synthesis and cell wall softening enzyme activity in tomato fruit during ripening by ethanol.

The exposure of mature-green tomato fruits (Lycopersicon esculentum Mill. cv. Bombay) to ethanol vapour at 0, 2.5, 5, 10, 20 or 25 ml/2.5 kg fruit/5 liter volume of jar delayed the ripening of fruits. The delay was found to be concentration dependent. The most effective concentration of ethanol was found to be 20 ml/2.5 kg fruit in delaying ripening. Ethanol reduced respiration rate, ethylene production and delayed the climacteric peak. The regulation of tomato fruit ripening was attributed to delay in fruit softening caused by reduced activities of enzymes like polygalacturonase (PG) and pectin methyl esterase (PME). The possible role of PG and PME activity in delaying ripening and maintaining fruit softness is also discussed.

Dehiscence‐related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus. The A. thaliana PG promoter was fused to β‐glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG‐barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed.

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

Commercialization of a tomato with an antisense polygalacturonase gene: The FLAVR SAVR™ tomato story

The FLAVR SAVR™ tomato was developed through the use of antisense RNA to regulate the expression of the enzyme polygalacturonase (PG) in ripening tomato fruit. This enzyme is one of the most abundant proteins in ripe tomato fruit and has long been thought to be responsible for softening in ripe tomatoes. The FLAVR SAVR™ tomato is the first genetically engineered whole food to be sold in commerce. The history of the development of this product is discussed beginning with the results of theoriginal antisense work (including conclusions regarding the role of PG in ripe tomatoes) and will be followed by a description of the regulatory and food safety assessment. Finally, the development of an operating business to produce, market and distribute a genetically engineered whole food product is discussed.

Role of Polygalacturonase in Bean Leaf Abscission

The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed.

The role of polygalacturonase in root-hair invasion by nodule bacteria.

SUMMARY: The production of polygalacturonase in associations of nodule bacteria and seedlings of leguminous plants was investigated. Plants and bacteria were combined in the following ways: (a) different plant species were combined with infective strains (isolated from the same cross inoculation group) as well as non-infective ones (from foreign groups); (b) host plant species with different susceptibilities were combined with the same bacterial strain; (c) bacterial strains with different in-fectivities, as measured by the number of infection sites, were combined with the same host species; (d) clover strains which had lost their infectivity, as well as transformed and again infective subcultures of these strains, were tested on clover plants. The results indicate that infection of the seedlings was strongly correlated with the production of polygalacturonase. The conclusion is drawn that polygalacturonase plays an important part in the infection process. This function is thought to be a weakening of the cell wall of the root hair which would facilitate the bacterial invasion. The possible role also of indolyl-3-acetic acid in the infection process is discussed.