Total Polygalacturonase Activity Research Articles

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity invitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

POLYGALACTURONASE INVOLVED IN EXPANSION1 Functions in Cell Elongation and Flower Development in Arabidopsis

Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.

Extraction and characterization of polygalacturonase of Fomes sclerodermeus produced by solid-state fermentation.

Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 degrees C and had optimum activity at pH 5 and 50 degrees C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.

The polygalacturonase

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG down-regulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria × ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening.

The strawberry (Fragaria x ananassa 'Chandler') fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.

Polygalacturonase activity and expression of related genes during ripening of strawberry cultivars with contrasting fruit firmness

Fleshy fruits soften during ripening mainly as a consequence of the catabolism of cell wall components. In strawberry ( Fragaria × ananassa Duch), the depolymerization and solubilization of pectins increase during ripening and contribute to fruit softening. In the present paper, we report the cloning and expression analysis of two polygalacturonase (PG) putative cDNAs: FaPG1 and T-PG. The former seems to be the same sequence of previously reported PG in strawberry, while T-PG cDNA has a deletion of 85 bp that cause a frame shift and would produce an inactive protein. Measurement of total PG activity and expression of FaPG1 and T-PG were performed in strawberry cultivars with contrasting softening rates. The softest cultivar (Toyonaka) showed the higher total PG activity in all ripening stages analyzed. The analysis by RT-PCR revealed that both genes express in the three cultivars, though the expression pattern was different. In the firmer cultivars (Selva and Camarosa) the expression of T-PG was considerably higher than the expression of FaPG1, while the opposite occurred in the softest cultivar (Toyonaka). Therefore, the higher PG activity detected in Toyonaka correlates with the enhanced expression of FaPG1 gene, while the low PG activity found in the firm cultivars correlates with a higher expression of T-PG, a gene that could encode a truncated protein without PG activity. The influence of auxins on both the expression of PG genes and the total PG activity during strawberry fruit ripening was also analyzed.

The effects of manothermosonication on tomato pectic enzymes and tomato paste rheological properties

The effect of manothermosonication (MTS), the simultaneous application of heat and high energy ultrasound waves under moderate pressure, on tomato juice rheology and on tomato pectinmethylesterase (PME) and polygalacturonase (PG) activity in tomato juice was examined. Tomato juice was subjected to MTS treatments (20 kHz, 2 kg pressure, 117 μm amplitude and 70 °C), or to control thermal treatments (TT), for 1 min. Flow curves, yield stress and apparent viscosity were measured on both kind of samples. Apparent viscosity of MTS samples were 1.6-fold higher than that of TT samples. Yield stress values were also 2.2-fold higher in MTS than in TT samples. Flow curve values were fitted to the power law equation; MTS samples showed about 1.9-fold higher consistency values and lower flow indexes, n=0.190 vs. n=0.126, than TT samples. MTS samples retained about 20% more serum than TT samples after centrifugation. TT inactivated about 38% of the initial PME activity whereas PME activity was undetectable in MTS-treated tomato juice. TT left PG unaffected whereas MTS treatments inactivated 62% of total PG activity. These results suggest, that MTS could be an useful technology to obtain high viscosity and consistency tomato juice.

A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger.

A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion-exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8-4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The K(m) values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur-containing amino acids. Both enzymes were rich in beta-structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, T(m), of the two enzymes was found to be 43 degrees C for PG II and 46 degrees C for PG IV.

Fusarium solani endo-polygalacturonase from decayed muskmelon fruit: purification and characterization

Fusarium solani is one of the more important fungal pathogens involved in pre- and post-harvest decay of muskmelon fruit. Production of polygalacturonase (PG), by F. solani was studied in vitro and in vivo . The fungus produced at least 14 PG isozymes with pIs of 4.5 to 9.5 in shake culture using pectin as the sole carbon source. When glucose and pectin were used in combination as the carbon source, total PG activity decreased substantially as compared to pectin alone, suggesting that glucose may suppress PG production in vitro . Only one PG isozyme, designated as PG1, was detected in extracts from infected fruit tissue. PG1 from decayed fruit was purified to homogeneity by protein extraction, ultrafiltration, gel filtration chromatography, and cation exchange chromatography. The molecular weight of PG1 was estimated at 38 kDa based on SDS-PAGE with a pI of 9.5 according to IEF-PAGE. PG1 exhibited only endo-PG activity based on viscosity reduction and thin layer chromatography analysis of products released by enzymatic action. The optimum pH for PG1 activity was 6. TheKm and Vmaxof PG1 using polygalacturonic acid as the substrate were 1.34 mg ml-1and 0.30 unit μg protein-1, respectively. PG1 effectively macerated fruit tissue which suggests that it may play an important role in decay of muskmelon fruit caused by F. solani .

A major gene controlling pathogenicity inBotryotinia fuckeliana(Botrytis cinerea)

Non-aggressive mutants were isolated from a single-ascospore strain ofBotryotinia fuckelianafollowing ultraviolet and chemical mutagenesis of conidia and screening on French bean leaves (Phaseolus vulgaris). Crosses with reference strain SAS56 revealed one ultraviolet induced mutant (Mp97) in which the non-aggressive phenotype segregated 1:1 indicating control by a single gene with a major effect on pathogenicity. Further crosses showed that this gene, designatedPat1, is not linked toMbc1(benzimidazole resistance),Daf1(dicarboximide resistance),nit1(nitrate non-utilising), orSel1(sodium selenate resistance). The pathogenicity ofPat1strains was compared with various wild-type strains. Mutant strains produced small, restricted lesions on leaves of French bean and soybean (Glycine max) and slowly spreading lesions on rose (Rosaspp.) flowers. The mutant was essentially non-pathogenic on tomato stems at 20 and 25 °C but produced a few invasive infections at 10 and 15 °C. Tests of radial growth rate, sporulation and sclerotial production showed thatPat1strains exhibit no gross unfitness or unusual nutrient requirement. Althoughin vivostudies showed substantially less total polygalacturonase activity inPat1lesions compared to those produced by wild-type strains, differences in polygalacturonase isozyme profiles were not correlated with the presence ofPat1.Pat1strains showed relatively low acid production in pH indicator medium. Microscopic examination showed that lesions induced by the Mp97 mutant on French and soybean leaves (4 days old), but not by its parent, were surrounded by a distinct staining zone of mesophyll cells suggesting a difference in host response. However, no differences in phytoalexin induction were found in a soybean assay.

Effects of chilling on tomato fruit texture

The effects of chilling on tomato (Lycopersicon esculentum Mill cv. Caruso) texture were investigated using fruit stored at 22°C (nonchilled) or 5°C (chilled) for 28 days. or at 5°C for 15 days before transfer to 22°C to facilitate ripening during and additional 13 days (prechilled). Prechilled fruit exhibited symptoms of slight chilling injury, i.e. development of mealiness, accelerated softening relative to that of nonchilled fruit and nonuniform surface colour development. The firmness of all fruit decreased during ripening and chilled storage when measured by flat plate compression and puncture, especially during the early stages of ripening of nonchilled and prechilled fruit. The compression firmness of pericarp tissue similarly decreased during ripening of nonchilled and prechilled fruit, but was maintained during chilling. Total moisture content (ca 94%) of tissue, uronide content (32‐35% w/w) and extracted β‐galactosidase activity did not differ significantly (P > 0.05) among fruit during ripening and chilled storage. The degree of uronide methyl esterification in ethanol‐insoluble solids prepared from pericarp tissue (EIS) was relatively low for all fruit. i.e. <40%. EIS from which greater levels of pectinesterase were extracted (i.e. nonchilled>chilled>prechilled) exhibited decreased levels of uronide methyl esterification. Markedly elevated levels of β‐glucosidase activity were extracted from prechilled EIS. Total polygalacturonase activity (mainly as PGI) and autolysis of enzyme‐extracted EIS were inversely correlated (P≤ 0.05) only with the loss of nonchilled fruit and tissue firmness and prechilled fruit firmness. Results suggest a possible role for β‐glucosidase in textural changes of prechilled fruit and tissue (e.g. loss of firmness, development of mealiness) and also implicate loss of skin strength in the softening of whole fruit during chilling.

Polygalacturonases Associated with Infection of Valencia Orange byPenicillium italicum

Three forms of polygalacturonase (PG), designated PG-I, PG-II, and PG-III, were purified to homogeneity from culture filtrate of Penicillium italicum. PG-I was characterized as an exoenzyme, whereas PG-II and PG-III wee characterized as endoenzymes. All three enzymes were present in orange peel infected by P. italicum. Exo-PG-I was the predominant enzyme in infected tissue and constituted 69% of the total PG activity as compared with 4% in the culture filtrate. Endo-PG-II and endo-PG-III constituted only 24 and 7%, respectively, of the activity in the infected tissues, as compared with 71 and 25% in culture filtrate (...)

Regulation by carbohydrates of the sequential in vitro production of pectic enzymes by Botrytis cinerea

Cultures of Botrytis cinerea in a basal salt medium supplemented with different pectin-related polysaccharides (French bean cell walls; citrus pectin; sodium polygalacturonate) as the only carbon source were examined daily for polygalacturonase activity, type of pectic enzymes present, and mycelial growth. Total polygalacturonase activity and number of enzymes detectable were influenced by type and concentration of the substrate and by the conidial concentration at which the cultures were started. A consistent sequence in the production of pectic enzymes was found. The polygalacturonase PG2 was always the first enzyme present in the culture filtrates and was followed by a number of polygalacturonase and pectinesterase isoenzymes. PG2 was also found in ungerminated conidia. Its production is the expression of a constitutive gene as it was independent of the presence of the substrate and strictly correlated with fungal growth. D-Galacturonic acid at 2 mM induced the production of some of the pectic enzymes. At 10 mM and above, however, it repressed PG2 and the subsequent production of the whole pectic isoenzyme complex, indicating a feedback repression. The results suggest that the pectic isoenzymes produced by B. cinerea constitute a coordinated catabolic pathway for the complete degradation of pectic polysaccharides.

Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains endo-PG I, endo-PG II and pectin esterase (PE)(with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.