Cloning and Expression of a cDNA Encoding Endopolygalacturonase from Feicheng Peach (Prunus persica)

Polygalacturonase (PG,EC3.2.1.15) is the key cell wall hydrolase in fruit ripening. The identification and characterization of a full length cDNA (pMT18) encoding for PG from Feicheng peach (Prunus persica (L.) Batsch cv. Feicheng) is described. The pMT18 clone is 1188 bp in length, with an open reading frame of 393 amino acids. The homology and phylogenetic analyses indicate a remarkable similarity between peach PG and other ripening related PG. And seven consensus sequences have revealed in peach PG compared to the PG from other plants. However, the profound divergence with other PG and the unique structure features suggest that peach PG probably belongs to a new evolutionary class. In RT PCR analysis, pMT18 related RNA was undetectable in leaves, and was much abundant in ripe fruits. The ripening specific expression pattern of this cDNA will be useful in investigating the roles of PG in fruit ripening and developing a transgenic peach with the improved post harvesting quality in the future.

Effect of Ethylene on Polygalacturonase,Lipoxygenase and Expansin in Ripening of Tomato Fruits

Fruit ripening is a complex process and is regulated by many factors. Ethylene and polygalacturonase （PG）, lipoxygenase （LOX）, expansin （EXP） are all critical regulating factors in fruit ripening and softening process. With antisense ACS tomato, Nr mutant tomato and cultivated tomato as materials, Northern blot hybridization showed that PG, LeEXP1 and LOXexpressed differently in different parts of cultivated tomato fruit during ripening, which was related to fruit ripening. The ripening process of columella and radial pericarp was faster than pericarp. In both Nr mutant and antisense ACS transgenic tomato fruit, expression levels ofPG, LeEXPI and LOXwere generally lower than those in cultivated fruit but still related to fruit ripening. The expression levels ofPG, LeEXP1 and LOX increased in the mature green tomato fruits after 0.5 h treatment with ethylene （100 μL/L）. These results indicate that gene expression ofPG, LeEXP1 and LOXwere positively regulated by ethylene. The time and cumulative effect of the concentration exists in the expression of PG regulated by ethylene. The regulation of LOX expression mainly depended on the fruit development after great amount of ethylene was produced. PG played a major role in ripening and softening of tomato fruit, and cooperated with the regulation of EXP and LOX.

Cloning and Sequence Analysis of Polygalacturonase(PG) Promoter in Tomato

In this study, 1 500 bp PG gene promoter was amplified from leaves of tomato cultivar ＇ Zhongshu No. 4＇. The bioinformatic analysis of PG promoter sequences was conducted. PG promoter elements were predicted and analyzed by PLANTCARE and PLACE. The results showed that tomato PG promoter contained multiple c/s-acting regulatory elements such as typical basic elements TATA-Box and CAAT-Box, light responsive elements 3-AF1 binding site, ATl-motif, ATCT- motif, Box 4, Box I, GA-motif, GTl-motif, Spl and MRE, heat stress-responsive element HSE, ethylene-responsive element ERE, meristem-specifie regulatory element CCGTCC-box, endosperm expression-related regulatory elements GCN4_motif and Skn-l_motif, defense and stress responsiveness element TC-rich repeats, and circadian control-related element circadian, indicating that the expression of tomato PG gene is related to light, temperature, hormone, stress and other factors. This study laid the foundation for subsequent research about regulation of PG gene expression in plants.

Characterization of an Exopolygalacturonase from Leucoagaricus gongylophorus, the Symbiotic Fungus of Atta sexdens

The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of KM, Vmax and kcat were 0.65 mg·mL-1, 1800 μmol·min-1·mg-1 and 35.97 s-1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0;otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.

Application and optimization of solid-state fermentation process for enhancing polygalacturonase production by Penicillium expansum

Penicillium expansum 3.5425 was applied in solid-state fermentation(SSF)of agricultural wastes for polygalacturonase biosynthesis.Among various carbon additives,apple pomace was most suitable for the biosynthesis of polygalacturonase(1440.57 U/g).Optimization of medium parameters using rotational orthogonal design(ROD)experiment combined with optimal fermentation conditions resulted in a 2.72-fold increase in the polygalacturonase production.By using ammonium sulphate precipitation,ion-exchange and gel-permeation chromatography,the polygalacturonase produced by P.expansum 3.5425 was finally purified which had specific activity of 19269 U/mg and molecular weight of 30 kDa.The enzyme was remarkably active in the pH range of 3-5 and at 50℃,which makes it more acceptable in the industrial application.Besides,partially purified polygalacturonase(875.15 U/mL)was used for apple juice clarification and the clarity at 0.4 mL/kg was maximum,which reveals a great potential of polygalacturonase in food industry.

Identification and Phylogenetic Analysis of the POLYGALACTURONASE Gene Family in Apple

In this study, a total of 85 apple polygalacturonase genes were characterized and clustered into seven groups based on the Malus × domestica whole-genome sequence. These genes coded for proteins containing 176–1 125 amino acids with isoelectric points ranging from 4.68 to 9.58.The predicted Md PG genes were distributed on all chromosomes except the 14 th. We then systematically analyzed conserved Md PG protein motifs and the structures of Md PG genes. We identified Md PG proteins containing four conserved motifs that are widely found in different PG proteins.Additionally, we found that Md PG75 was the largest gene, encompassing 18 exons. Finally, we systematically analyzed the functional connection network of Md PG proteins and predicted the functions of related Md PG genes before undertaking a preliminary validation. Overall, we have described the genome-wide identification and analysis of the apple PG gene family.

Endo-Glycanhydrolases Activities in Artemisia sphaerocephala (Asteraceae) Mucilaginous Achene Germination Process

对白沙蒿 (ArtemisiasphaerocephahaKrasch .)种子萌发不同阶段的种胚提取物中几种可能降解果皮外层粘液物质的多糖内切酶进行了检测。研究结果表明 :多聚半乳糖醛酸酶在干燥胚中已存在并具较高的活性 ,但其活性随着种子吸涨及萌发而降低。此酶并不释放到种胚外。β 甘露聚糖内切酶随着种子的吸涨而增加并被萌发的种胚释放到种子外。纤维素酶在种子萌发的过程中不表现出活性。提取物中的所有多糖内切酶都无法降解果皮外层的粘液物质。

Combinatorial Enzyme Approach to Produce Oligosaccharides of Diverse Structures for Functional Screen

Combinatorial chemistry has been a focus of research activity in modern drug discovery and biotechnology. It is a concept by which a vast library of molecular diversity is synthesized and screened for target properties. This report is to illustrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Citrus pectin was subjected to combinatorial enzyme digestion to create libraries of pectic oligosaccharides with diverse structural variants. Repeated cycles of fractionation and screening resulted in the isolation and identification of an active oligoGalA species with antimicrobial activity.

Physical, Chemical and Biochemical Changes of Sweetsop （Annona squamosa L.） and Golden Apple （Spondias citherea Sonner） Fruits during Ripening

This study aimed at the physical, chemical and biochemical changes during ripening of Sweetsop （Annona squamosa L.） and Golden Apple （Spondias citherea Sonner） fruits during ripening as important features to better understand their postharvest handling. It was carried out physical analysis such as firmness and chemical analysis such as total chlorophyll, total carotenoids, soluble solids, pectins and titrable acidity and biochemical analysis such as pectin methyl esterase, polygalacturonase, cellulase, and peroxidase and polyphenoloxidase activities in crude extract. Fruits were harvested at different stages of ripening. Experimental design was completely randomized and was carried out analysis of variance and Tukey tests, Total chlorophyll was decreasing in later stages of ripening, total soluble solid contents increased as the fruits ripen, while the acidity expressed percentage of citric acid decreased during fruits ripening. The loss of firmness and soluble solids content increased as the fruit get ripped stage, while the content of pectin decreased. Activity was observed for pectin methyl esterase and polygalacturonase enzymes during all stages of maturation, presenting the highest activity for both enzymes in the mature state. No cellulase activity detected at any stage during the ripening of these fruits. Activity of the enzyme polyphenoloxidase and peroxidase, associated with pulp browning was higher in the last stages of ripening of these fruits. Physical, chemical and biochemical patterns during ripening were different according to fruit species suggesting differential postharvest handling requirements.

Ethylene†and fruit softening

This review is concerned with the mechanisms controlling fruit softening.Master genetic regulators switch on the ripening programme and the regulatory pathway branches downstream,with separate controls for distinct quality attributes such as colour,flavour,texture,and aroma.Ethylene plays a critical role as a ripening hormone and is implicated in controlling different facets of ripening,including texture change,acting through a range of transcriptional regulators,and this signalling can be blocked using 1-methylcyclopropene.A battery of at least seven cell-wall-modifying enzymes,most of which are synthesized de novo during ripening,cause major alterations in the structure and composition of the cell wall components and contribute to the softening process.Significant differences between fruits may be related to the precise structure and composition of their cell walls and the enzymes recruited to the ripening programme during evolution.Attempts to slow texture change and reduce fruit spoilage by delaying the entire ripening process can often affect negatively other aspects of quality,and low temperatures,in particular,can have deleterious effects on texture change.Gene silencing has been used to probe the function of individual genes involved in different aspects of ripening,including colour,flavour,ethylene synthesis,and particularly texture change.The picture that emerges is that softening is a multi-genic trait,with some genes making a more important contribution than others.In future,it may be possible to control texture genetically to produce fruits more suitable for our needs.