Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Overexpression and Biochemical Characterization of an Endo-α-1,4-polygalacturonase from Aspergillus nidulans in Pichia pastoris.

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-α-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from others. AnEPG overexpressed in Pichia pastoris was characterized. AnEPG showed the highest activity at pH 4.0, and exhibited moderate activity over a narrow pH range (pH 2.0-5.0) and superior stability in a wide pH range (pH 2.0-12.0). It displayed the highest activity at 60 °C, and retained >42.2% of maximum activity between 20 and 80 °C. It was stable below 40 °C and lost activity very quickly above 50 °C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the Km and kcat values of 8.3 mg/mL and 5640 µmol/min/mg, respectively. Ba2+ and Ni2+ enhanced activity by 12.2% and 9.4%, respectively, while Ca2+, Cu2+, and Mn2+ inhibited activity by 14.8%, 12.8%, and 10.2% separately. Analysis of hydrolysis products by AnEPG proved that AnEPG belongs to an endo-polygalacturonase. Modelled structure of AnEPG by I-TASSER showed structural characteristics of endo-polygalacturonases. This pectinase has great potential to be used in food industry and as feed additives.

Identification and Expression Analysis of Polygalacturonase Family Members during Peach Fruit Softening.

Polygalacturonase (PG) is an important hydrolytic enzyme involved in pectin degradation during fruit softening. However, the roles of PG family members in fruit softening remain unclear. We identified 45 PpPG genes in the peach genome which are clustered into six subclasses. PpPGs consist of four to nine exons and three to eight introns, and the exon/intron structure is basically conserved in all but subclass E. Only 16 PpPG genes were expressed in ripening fruit, and their expression profiles were analyzed during storage in two peach cultivars with different softening characteristics. Eight PGs (PpPG1, -10, -12, -13, -15, -23, -21, and -22) in fast-softening "Qian Jian Bai" (QJB) fruit and three PGs (PpPG15, -21, and -22) in slow-softening "Qin Wang" (QW) fruit exhibited softening-associated patterns; which also were affected by ethylene treatment. Our results suggest that the different softening characters in QW and QJB fruit is related to the amount of PG members. While keeping relatively lower levels during QW fruit softening, the expression of six PGs (PpPG1, -10, -12, -11, -14, and -35) rapidly induced by ethylene. PpPG24, -25 and -38 may not be involved in softening of peach fruit.

Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

Optimization, purification and characterization of pectinases from pectinolytic strain, Aspergillus foetidus MTCC 10559.

A strain (MPUAT-2), isolated from coconut hull and identified as Aspergillus foetidus MTCC 10559, was used for pectinase production. Optimum pectinase production was obtained at pH 8.0 and temperature 35 degrees C under static conditions in submerged fermentation after 5 days of incubation. Orange peel, a byproduct of fruit industry, was used as a sole carbon source (3% w/v) to produce high pectinase, thus making the process cost effective. The culture filtrate was analyzed for pectin methyl esterase (PME) and endopolygalacturonase (endo-PG) enzymes. The enzymes, PME and endo-PG were purified using ammonium sulphate precipitation and molecular exclusion chromatography (Sephadex G-75) with corresponding recovery of 39.3 and 44.3%. The partially purified enzymes were also characterized for their kinetic properties.

Cold active pectinases: advancing the food industry to the next generation.

Pectinase has been an integral part of commercial food processing, where it is used for degradation of pectin and facilitates different processing steps such as liquefaction, clarification and juice extraction. The industry currently uses pectinases from mesophilic or thermophilic microorganisms which are well established, but recently, there has been is a new trend in the food industry to adopt low-temperature processing. This trend is due to the potential economic and environmental advantages which the industry envisages. In order to achieve this change, an alternative for the existing pectinases, which are mostly mesophilic and temperature-dependent, must be identified, which can function efficiently at low temperatures. Psychrophilic pectinases derived from cold-adapted microorganisms, are known to function at low to freezing temperatures and may be an alternative to address the problem. Psychrophilic pectinases can be obtained from the vast microflora inhabiting various cold regions on earth such as oceans, Polar Regions, snow-covered mountains, and glaciers. This article is intended to study the advantages of cold active pectinases, its sources, and the current state of the research.

ps-2, the gene responsible for functional sterility in tomato, due to non-dehiscent anthers, is the result of a mutation in a novel polygalacturonase gene.

The recessive mutation ps-2, which appeared spontaneously in tomato, confers functional male sterility due to non-dehiscent anthers. In this study, we isolated and characterized the PS-2 gene. A single nucleotide mutation in a novel tomato polygalacturonase gene is responsible for the ps-2 phenotype. The mutation in ps-2 is responsible for an alternative splicing during maturation of the pre-mRNA, which leads to an aberrant mRNA. Differentiation between ps-2 and wild type (PS-2) anthers only appears in the final developmental stage in which the stomium remains closed in the mutant. To our knowledge, this is the first functional sterility gene isolated in the Solanaceae family. The specific expression of the Arabidopsis homolog of PS-2 in the anther dehiscence zone suggests a conserved mode of action over the plant kingdom, which means that the repression of PS-2 homologs may be a potential way to introduce functional sterility in other species.

Evolutionary analysis of endopolygalacturonase-encoding genes of Botrytis cinerea.

Sequence analysis of five of the six endopolygalacturonase-encoding genes (Bcpg1, Bcpg2, Bcpg3, Bcpg4, Bcpg5) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald-Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2, can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.

Pectin methylesterase and polygalacturonase in the developing grape skin.

Ripening of grape (Vitis vinifera L.) berry immediately precedes harvesting and the evolution of the skin tissue is important as it contains the key compounds for wine quality. Grape softening is thought to result from extensive cell wall modifications that occur during ripening. These modifications result from the activity of different cell wall-modifying enzymes. Two of the most significant pectin-degrading enzymes are pectin methylesterase (EC 3.1.1.11) and polygalacturonase (EC 3.2.1.15). In this work, the activities of both enzymes were monitored in skin tissue throughout berry development. Pectin methylesterase activity was present before the onset of veraison and increased during skin maturation. No polygalacturonase activity could be detected. The accumulation of mRNA encoding a pectin methylesterase and two polygalacturonase isoforms was examined using RT-PCR. Transcripts for pectin methylesterase were present in all stages analyzed with a maximal accumulation at the end of color change. Accumulation of VvPG1 transcript was closely correlated with berry softening, and expression of this gene was markedly increased during the color change. VvPG2 mRNA accumulation began before veraison and was low during skin ripening. A phylogenic analysis showed that this gene is classified in a different group than VvPG1. These findings suggest that both genes are associated with different mechanisms during skin development. VvPG1, in particular, is most likely to play a role in skin softening and VvPG2 in triggering the ripening process.

Gene structure dynamics and divergence of the polygalacturonase gene family of plants and fungus.

Whole copies of the polygalacturonase (PG) genes from rice (Oryza sativa subsp. japonica) and a filamentous fungus (Aspergillus oryzae) were isolated. The orthologs of the rice PGs were also retrieved from other plant species. The 106 plant PGs analyzed were divided into 5 clades, A, B, C, D, and E. The fungus PGs were classified into 3 clades, of which one formed a loose cluster with clade E of the plant PGs. Four domain motifs (I, II, III, IV) were identified in all PGs. Motifs II and III were split by introns such as G/DDC and CGPGHGIS/IGSLG, respectively. In plant PGs there were 446 introns in total and 3.98 introns per gene. Intron phase distribution was 65.5% for phase 0, 19.7% for phase 1, and 14.8% for phase 2 in plant PGs. In the PGs of A. oryzae there were 37 introns of phase 0 (59.5%), phase 1 (24.3%), and phase 2 (16.2%), with 2.47 introns per gene. The 5 clades of plant PGs were divided into 3 basic gene structure lineages. Intron positions and phases were conserved among the PGs in the first 2 lineages. The third lineage consisted of PGs of clade E, which also carried highly conserved introns at different positions from other PGs. Intron positions were not as highly conserved in fungus PGs as in plant PGs. The introns in the current PGs have been present since before the divergence of monocots from dicots. The results obtained show that differential losses of introns created gene diversity, which was followed by segmental and tandem duplication in plant PGs.

Influence of beta-subunit on thermal and high-pressure process stability of tomato polygalacturonase.

Polygalacturonase (PG; E.C. 3.2.1.15) was extracted from tomato fruit and purified by cation-exchange chromatography. Two peaks containing PG activity were detected: the first denotes a thermolabile PG fraction (PG2) and the second a thermostable fraction (PG1). PG1 is a dimer of PG2 and a heat-stable protein called the beta-subunit. In contrast to its resistance to heat, PG is easily inactivated at elevated pressure. Although the thermal stability of purified tomato PG1 and PG2 is distinctly different, they show an identical pressure stability. To gain further insight into the thermal and pressure stability of both PG isoenzymes, the in vitro recombination of PG2 and beta-subunit was studied. After severe heat (up to 140 degrees C for 5 min) and pressure (up to 800 MPa for 15 min) treatments, the residual fractions containing the beta-subunit were able to convert PG2 into the heat-stable PG1, showing the extreme thermal and pressure stability of the beta-subunit. PG1 was detected in heat-treated tomato juice and, to a lesser extent, in tomato pieces. In contrast, as was the case for purified PG, no pressure-stable fraction was observed when tomato juice and pieces were treated under pressure. These data clearly show the differing behavior of the PG1-PG2-beta-subunit system under thermal and high-pressure treatments and offer the possibility of inactivating tomato PG using high pressure without the need for high temperatures.

A polygalacturonase of animal origin isolated from the root-knot nematode Meloidogyne incognita.

The first animal polygalacturonase (PG, EC 2.1.15) encoding cDNA, Mi-pg-1, was cloned from the plant parasitic nematode Meloidogyne incognita. The enzymatic activity of MI-PG-1 was confirmed after heterologous expression in Escherichia coli. The presence of a predicted signal peptide on the MI-PG-1 sequence together with the specific localization of the transcripts of the Mi-pg-1 gene in the oesophageal glands of infective juveniles imply that MI-PG-1 could be secreted into plant tissues. The potential role of MI-PG-1 in parasitism is discussed.

Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.