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In the last decade, the application of non-Saccharomyces yeasts in oenology as a natural tool to obtain wine diversification and higher quality has aroused great interest. In this work, three Metschnikowia pulcherrima strains, isolated from a vineyard, were characterised through the evaluation of their main oenological properties, antimicrobial activity, and specific enzymatic activities (β-glucosidase, β-lyase, polygalacturonase, and protease). The M. pulcherrima strains did not produce any inhibition against Saccharomyces cerevisiae, while they were able to exert an antimicrobial action against some unwanted bacteria and yeasts frequently present in grape must and potentially causing the alteration of wines. After this preliminary screening, M. pulcherrima AS3C1 has been selected to be used in the winemaking of red grape Vitis vinifera cv. Aglianico on a pilot scale. The effect of the sequential inoculation of M. pulcherrima AS3C1 with a commercial strain of S. cerevisiae was verified using for comparison a single inoculum with S. cerevisiae and a spontaneous fermentation. Our results showed a higher concentration of anthocyanins and catechins in wines obtained by the sequential inoculation of M. pulcherrima AS3C1 and S. cerevisiae. On the basis of the data obtained, M. pulcherrima AS3C1 possesses an enzymatic profile and some oenological properties that could contribute positively to the definition of the chemical composition of wines, suggesting its possible use for red winemaking processes. [ABSTRACT FROM AUTHOR]

The main objective of this study was to monitor apricot development and ripening through gene expression analysis of key candidate genes using the RT-qPCR technique. Eight apricot cultivars were selected to analyze phenological and genetic patterns from pre-ripening stages through to postharvest. In addition, 19 selected genes were analyzed in the contrasting cultivars 'Cebas Red' and 'Rojo Pasión' in different stages (two preharvest stages S1 and S2, one harvest stage S3, and two postharvest stages S4 and S5). This pool of genes included genes related to fruit growth and ripening, genes associated with fruit color, and genes linked to the fruit's nutraceutical aspects. Among the studied genes, Polygalacturonase (PG), Pectin methylesterase (PME), Aminocyclopropane-1-carboxylate synthase (ACS), and Myo-inositol-1-phosphate synthase (INO1) were directly related to fruit maturation and quality. Significant differential expression was observed between the cultivars, which correlated with variations in firmness, shelf life, and sensory characteristics of the apricots. 'Rojo Pasión' displayed high levels of PG, associated with rapid maturation and shorter postharvest shelf life, whereas 'Cebas Red' exhibited lower levels of this gene, resulting in greater firmness and extended shelf life. Genes CCD4, CRTZ, and ZDS, related to carotenoids, showed varied expression patterns during growth and postharvest stages, with higher levels in 'Rojo Pasión'. On the other hand, Sucrose synthase (SUSY) and Lipoxygenase (LOX2) were prominent during the postharvest and growth stages, respectively. Additionally, GDP-L-galactose phosphorylase (VTC2_5) was linked to better postharvest performance. This research provides valuable insights for future breeding initiatives aimed at enhancing the quality and sustainability of apricot cultivation. [ABSTRACT FROM AUTHOR]

Fruit softening is a prominent attribute governing both longevity on shelves and commercial worth. Polygalacturonase (PG) plays a major role in strawberry fruit softening. However, the PG gene family in strawberry has not been comprehensively analyzed. In this study, 75 FaPG genes were identified in the octoploid strawberry genome, which were classified into three groups according to phylogenetic analysis. Subcellular localization prediction indicated that FaPGs are mostly localized to the plasma membrane, cytoplasm, and chloroplasts. Moreover, the expression of FaPGs during strawberry development and ripening of 'Benihoppe' and its softer mutant was estimated. The results showed that among all 75 FaPGs, most genes exhibited low expression across developmental stages, while two group c members (FxaC_21g15770 and FxaC_20g05360) and one group b member, FxaC_19g05040, displayed relatively higher and gradual increases in their expression trends during strawberry ripening and softening. FxaC_21g15770 was selected for subsequent silencing to validate its role in strawberry softening due to the fact that it exhibited the highest and most changed expression level across different developmental stages in 'Benihoppe' and its mutant. Silencing FxaC_21g15770 could significantly improve strawberry fruit firmness without affecting fruit color, soluble solids, cellulose, and hemicellulose. Conversely, silencing FxaC_21g15770 could significantly suppress the expression of other genes related to pectin degradation such as FaPG-like, FaPL, FaPME, FaCX, FaCel, FaGlu, FaXET, and FaEG. These findings provide basic information on the FaPG gene family for further functional research and indicate that FxaC_21g15770 plays a vital role in strawberry fruit softening. [ABSTRACT FROM AUTHOR]

The addition of pectinase enzymes during the maceration stage of grape skins in order to improve the extraction yields and color of red wines is a common practice in many wineries. The objective of this work was to study in depth the changes that occurred in the aminoacidic, volatile, and phenolic compositions of Cabernet Sauvignon wines fermented with a Saccharomyces cerevisiae strain genetically modified with the gene encoding for endopolygalacturonase (PGU1) in transcriptional fusion with the promoter of the phosphoglycerate kinase (PGK1) gene, both from S. cerevisiae origin. A higher yield extraction of wine was obtained in wines fermented with the modified strain (PW), increasing by around 6.1% compared to the control wine (CW). Moreover, there was a 40% decrease in the malic acid content in the PW, thus suggesting that this modified yeast could be investigated as a malic acid-reducing agent. There were slight differences in other aroma volatile compounds studied as well as in the phenolic content. However, there was a considerable increase in the amino acid content in the PW. [ABSTRACT FROM AUTHOR]

Mealy peaches are dry and flavorless, which reduces their consumer acceptance. A deeper understanding of the mechanism underlying mealiness is crucial to enhancing peach fruit quality. In this study, comparative profiling was conducted on CP13, CP14, CM, and RM peaches. Sensory evaluation indicated that CP13 and CM are non-mealy clingstone and freestone peaches, respectively, and CP14 and RM are mealy freestone peaches. Both CP13 and CP14, identified as stony hard (SH) peaches, exhibited minimal ethylene release, whereas CM and RM, identified as melting flesh (MF) peaches, released high amounts of ethylene during the ripening process. Scanning electron microscopy (SEM) microstructure observation indicated that cells in the flesh tissue of mealy peaches, CP14 (SH) and RM (MF), were intact and separated, with large intercellular spaces and irregular arrangements. The main factor that promotes mealiness is differences in pectin metabolism, which impact cell wall composition. The fluctuations in polygalacturonase (PG) and pectin methylesterase (PME) activity between mealy and non-mealy peaches were the main factor contributing to mealiness. However, the changes in cell wall metabolism that caused these fluctuations did not have a clear direction. Using transcriptome analysis and weighted gene co-expression network analysis (WGCNA), we were able to identify forty differentially expressed genes (DEGs) that are associated with mealy patterns. Among these DEGs, genes encoding PG were significantly upregulated in mealy peaches (CP14 and RM) compared to non-mealy peaches (CP13 and CM). PpPG1 was the main effector gene for mealiness, while PpPG2, PpEGase2, PpEXP1, PpEXP3, PpAGP2, PpIAA4, and PpABA2 were identified as candidate genes regulating peach mealiness. These findings provide a solid experimental basis for understanding the textual distinctions between mealy and non-mealy peaches. [ABSTRACT FROM AUTHOR]

The postharvest softening and corresponding quality deterioration of blueberry fruits are crucial factors that hinder long-distance sales and long-term storage. Cold plasma (CP) is an effective technology to solve this, but the specific mechanism of delaying fruit softening remains to be revealed. Here, this study found that CP significantly improved blueberry hardness. Physiological analysis showed that CP regulated the dynamic balance of reactive oxygen species (ROS) to maintain hardness by increasing antioxidant content and antioxidant enzyme activity, resulting in a 12.1% decrease in the H2O2 content. Transcriptome analysis revealed that CP inhibited the expression of cell wall degradation-related genes such as the pectin hydrolase gene and cellulase gene, but up-regulated the genes of the ROS-scavenging system. In addition, the resistance genes in the MAPK signaling pathway were also activated by CP in response to fruit ripening and softening and exhibited positive response characteristics. These results indicate that CP can effectively regulate the physiological characteristics of blueberries at a genetic level and delay the softening process, which is of great significance to the storage of blueberries. [ABSTRACT FROM AUTHOR]

Pectinase, a kind of hydrolase, mainly contains polygalacturonase, pectinase, and pectin lyase, which can hydrolyze pectin to generate galacturonide and is widely used in industry. At present, pectinase's activity is still relatively low. Hyperactive pectinase was produced with solid-phase fermentation and a tray bioreactor using the novel strain Aspergillus sp. CM96 in this study. This pectinase's activity can reach 17,000 U·g−1 after fermentation with a tray bioreactor, an increase of 86% compared to that obtained using flask liquid fermentation. The pectinase was purified and its characteristics were explored. Additionally, during pectinase fermentation, the activities of protease, glucanase, and cellulase were also determined to reach 7000, 8000, and 3000 U·g−1. The enzyme mixture was used to improve substrate digestion efficiency in 144 Soviet white pigs after adding a 0.05% cocktail enzyme for 38 days. The results showed that the average daily gain (ADG) increased by 139.41 ± 1.04 g·day−1, while the average daily feed intake (ADFI) and the feed conversion rate (FCR) decreased by 19.82 ± 1.64 g·day−1 and 0.07 ± 0.01 in 38 days, which indicated that the addition of hyperactive pectinase from the strain CM96 can increase nutrient digestibility and improve feed efficiency. [ABSTRACT FROM AUTHOR]

The high susceptibility of raspberries to softening restricts the development of the raspberry industry. The primary causes of fruit softening are the breakdown of components linked to the cell wall and the destruction of the cell wall structure itself. Polygalacturonase (PG), a key enzyme that catalyzes pectin degradation, plays a critical role in fruit softening. However, there are currently limited studies on the mechanism of PG genes in raspberry fruit softening. In this study, a PG gene, RiPG2, was isolated from raspberry (Rubus idaeus L.). 'Polka' fruits and tomato plants overexpressing RiPG2 were obtained by Agrobacterium tumefaciens-mediated leaf disc transformation to elucidate the role of RiPG2 in fruit softening. The total length of the RiPG2 gene is 1185 bp, and the gene encodes a total of 394 amino acids. The GFP fusion protein was expressed at the chloroplast under laser confocal microscopy, indicating that the RiPG2 protein is localized to the chloroplasts. Phenotypic analysis revealed that the fruit firmness of three strains was considerably less than that of controls, but PG enzyme activity was increased. Overexpression of RiPG2 altered the content of cell wall components, with an increase in water-soluble pectin (WSP) and ion-bound pectin (ISP) but a decrease in protopectin, cellulose, hemicellulose, and covalently bound pectin (CSP). In addition, RiPG2 positively regulated the expression of cell wall metabolism-related genes such as SlEXP1, SlTBG4, SlXTH5, and SlPL. These results suggest that the RiPG2 gene regulates the structure and composition of the cell wall and acts synergistically with other cell wall metabolism-related genes to promote fruit softening. This study provides a new candidate gene for molecular breeding to improve raspberry firmness. [ABSTRACT FROM AUTHOR]

Polygalacturonase (PG) is one of the largest families of hydrolytic enzymes in plants. It is involved in the breakdown of pectin in the plant cell wall and even contributes to peel cracks. Here, we characterize PGs and outline their expression profiles using the available reference genome and transcriptome of Akebia trifoliata. The average length and exon number of the 47 identified AktPGs, unevenly assigned on 14 chromosomes and two unassembled contigs, were 5399 bp and 7, respectively. The phylogenetic tree of 191 PGs, including 47, 57, 51, and 36 from A. trifoliata, Durio zibethinus, Actinidia chinensis, and Vitis vinifera, respectively, showed that AktPGs were distributed in all groups except group G and that 10 AktPGs in group E were older, while the remaining 37 AktPGs were younger. Evolutionarily, all AktPGs generally experienced whole-genome duplication (WGD)/segmental repeats and purifying selection. Additionally, the origin of conserved domain III was possibly associated with a histidine residue (H) substitute in motif 8. The results of both the phylogenetic tree and expression profiling indicated that five AktPGs, especially AktPG25, could be associated with the cracking process. Detailed information and data on the PG family are beneficial for further study of the postharvest biology of A. trifoliata. [ABSTRACT FROM AUTHOR]

Kiwifruits are often exposed to various temperature fluctuations (TFs) during postharvest transportation and storage. To evaluate the effect of TFs on the qualities of kiwifruits during storage, kiwifruits were stored at 2 °C, 2 °C or 5 °C (TF2 °C–5 °C, alternating every 12 h), 2 °C or 7 °C (TF2 °C–7 °C, alternating every 12 h) for 3 d before long time storage at 2 °C. Observations revealed that kiwifruits stored at a constant 2 °C showed the lowest loss of weight and vitamin C because of minimized ethylene production and respiratory rate compared with that of TF2 °C–5 °C and TF2 °C–7 °C. Moreover, the results of RT-qPCR verified that the expression levels of genes encoding polygalacturonase, β-galacturonidase, and pectin methylesterase were significantly increased by the treatment of TF. Hence, TF accelerated the degradation of cell walls, softening, translucency, and relative conductivity of the flesh of kiwifruits. In addition, the impact of TF2 °C–7 °C on kiwifruits was more significant relative to TF2 °C–5 °C. The present study provides a theoretical basis for kiwifruit during cold storage. [ABSTRACT FROM AUTHOR]

The aim of the study was to develop a biotechnological approach for the green recovery of chlorophyll from spinach, to be used as a natural food colorant. The plant matrix was characterized in terms of cell wall polysaccharide composition, and a tailored enzymatic mix based on cellulase (40%) xylanase (41%) and polygalacturonase (19%) was formulated. The process variables (temperature (°C), time (h), enzyme mix dose (U/g), zinc concentration (ppm), and buffer/substrate ratio (B/S)) and their interactions were studied by response surface methodology. The overlay plot made it possible to identify the process conditions (T: 25 °C, Zn: 150 ppm e B/S: 17.5, t: <2 h and enzyme mix dose between 12 and 45 U/g) to maximize the amount of chlorophyll, and concurrently, the quality of the green color of the extract. Finally, the novel colorant was applied in the production of a real food. [ABSTRACT FROM AUTHOR]

Cold plasma (CP) is a green and efficient preservation technology, but its effect on preventing the postharvest softening of fruits and vegetables is still unclear, and the effects of different CP intensities on the postharvest softening of winter jujubes (Ziziphus jujuba Mill. cv. Dongzao) have been little studied. In this study, we investigated the effects of different CP intensities (0 kV, 40 kV, and 80 kV) on the postharvest storage quality of winter jujubes and the activities of key enzymes related to softening during storage. The results showed that compared with the control group, the contents of firmness, total soluble solids (TSS), titratable acidity (TA), and vitamin C (VC) in the CP treatment group were higher and the respiratory intensity and weight loss rate were lower. In addition, CP treatment can also inhibit the degradation of protopectin and cellulose and the accumulation of soluble pectin. Moreover, CP treatment inhibited the activities of polygalacturonase (PG), pectin methyl esterase (PME), 3α-L-arabinoside (α-L-Af), 4β-galactosidase (β-gal), cellulase (Cx), and β-glucosidase (β-Glu). This shows that CP treatment has a positive effect on the preservation of winter jujubes, and the preservation effect of the 80 kV treatment was better than that of the 40 kV treatment. This provides a certain basis for cold plasma treatment in the preservation of winter jujubes. [ABSTRACT FROM AUTHOR]

Papaya (Carica papaya L.) is a climacteric fruit, and its quality will rapidly decrease after ripening. Hence, the storage life of its fruit is short. D-limonene is a terpene compound in citrus essential oil and has antibacterial and antioxidant properties. The addition of D-limonene in edible coating can delay volatilization, prevent microorganism and pathogen invasion, decrease water loss, inhibit softening, decrease gas exchange, and extend the storage life of fruits. In this study, 0.25%, 0.5%, and 1% D-limonene nanoemulsion coatings were used for post-harvest immersion treatment of "Tainung No. 2" papayas and its effects on appearance, chlorophyll content, respiration rate, ethylene production, pectin methylesterase, polygalacturonase activity, decay loss, firmness, total soluble solid, titratable acidity, ascorbic acid, and total plate count were investigated. After the papayas were treated with 0.5% D-limonene nanoemulsion coating, polygalacturonase and pectin methylesterase activities decreased, fruit firmness was maintained, and ascorbic acid content was high. On the last day of storage, polygalacturonase and pectin methylesterase activities were 0.01 and 0.02 U/kg FW lower than the control group, respectively; firmness was higher than the control group by 1.75 N, and ascorbic acid content was higher than the control group by 31.97 mg/100 g FW. On Day 2, the treatment showed delay in total soluble solid accumulation and chlorophyll degradation, consequently delaying the color change in fruits. The coating decreased decay loss by 40% on Day 2, decreased respiration rate by 97.0 mg CO2 kg−1 h−1, and ethylene production by 5.7 µL kg−1 h−1 on Day 2. Simultaneously, the coating decreased the total plate count and resulted in a good appearance. Fruits coated with a 1% D-limonene nanoemulsion coating showed defects in color change. In summary, 0.5% D-limonene nanoemulsion coating delayed "Tainung No. 2" papaya ripening and decreased microbial infection, consequently extending its storage life. [ABSTRACT FROM AUTHOR]

In this study, we aimed to isolate and phenotypically characterize non-Saccharomyces yeast strains (NSYSs) from the skin of aromatic (Italy) and non-aromatic (Negra Criolla) grapes from vineyards in Moquegua, Peru, typically used for the production of pisco. Our second objective was to characterize the volatile compounds and sensory attributes of pisco made from these grapes. Pichia terricola (56%), Metschnikowia pulcherrima (31%), and Naganishia vaughanmartiniae (13%) were the main NSYSs isolated from the skin of aromatic Italy grapes and identified; meanwhile, Vishniacozyma carnescens (50%), Vishniacozyma heimaeyensis (30%), and Aureobasidium pullulans (20%) were identified on the skin of the non-aromatic Negra Criolla grapes. These NSYSs showed different capacities in terms of carbohydrate fermentation, polygalacturonase activity, ethanol tolerance, sulphite production, and nitrogen consumption. Moreover, the pisco resulting from these varieties of grape had different volatile profiles. Terpene alcohols such as citronellol, geraniol, linalool, and nerol were found in pisco made from Italy grapes, while higher contents of 2-phenylacetate and ethyl esters were found in Negra Criolla Pisco. Intermediate levels of both 1-hexanol and bencyl alcohol were also found in all the pisco. Sensory analysis performed by a trained pisco tasting panel showed that citric, floral, alcohol, and syrup descriptors were more marked in Italy Pisco, while nuts, syrup, alcohol, and floral were the most intense attributes of Negra Criolla Pisco. These results will contribute to determining the potential of indigenous grape yeasts from the Moquegua region as fermentation starters to improve the typical sensory qualities of the pisco produced in this region, which deserves further study. [ABSTRACT FROM AUTHOR]

Plant height is one of the most crucial components of plant structure. However, due to its complexity, the genetic architecture of rice plant height has not been fully elucidated. In this study, we performed a genome-wide association study (GWAS) to determine rice plant height using 178 commercial rice varieties and identified 37 loci associated with rice plant height (LAPH). Among these loci, in LAPH2, we identified a polygalacturonase gene, OsPG3, which was genetically and functionally associated with rice plant height. The rice plant exhibits a super dwarf phenotype when the knockout of the OsPG3 gene occurs via CRISPR-Cas9 gene-editing technology. RNA-Seq analysis indicated that OsPG3 modulates the expression of genes involved in phytohormone metabolism and cell-wall-biosynthesis pathways. Our findings suggest that OsPG3 plays a vital role in controlling rice plant height by regulating cell wall biosynthesis. Given that rice architecture is one of the most critical phenotypes in rice breeding, OsPG3 has potential in rice's molecular design breeding toward an ideal plant height. [ABSTRACT FROM AUTHOR]

Extracted sugar beet press pulp (SBPP) is a promising agricultural residue for saccharification and further bioconversion. Combining solid-state fermentation of SBPP with engineered Aspergillus niger for enzyme production followed by hydrolysis of additionally added SBPP in the same bioreactor was studied to produce a sugar solution (hydrolysate) in a one-pot process. The initial aerobic solid-state fermentations were carried out in duplicate on non-milled, wet SBPP (moisture content of 72% (w/v)) with an A. niger strain engineered for constitutive pectinase production for 96 h, and this resulted in polygalacturonase activities of up to 256 U mL−1 in the wet media. Afterwards, water was added to the bioreactor, and the remaining solids were suspended by stirring to dissolve the hydrolytic enzymes. Metabolic activities of A. niger were inactivated by a N2-atmosphere and by increasing the temperature to 50 °C. High solid loads of milled SBPP were added to the stirred-tank reactor with a delay of 24 h to enable sugar yield calculations based on the compositional analysis of the SBPP used. The resulting final sugar concentrations of the hydrolysate after 166 h were 17 g L−1 d-glucose, 18.8 g L−1 l-arabinose, and 12.5 g L−1 d-galacturonic acid, corresponding to sugar yields of 98% d-glucose, 86% l-arabinose, and 50% d-galacturonic acid, respectively. Including the other sugars released during enzymatic hydrolysis in the one-pot process (d-xylose, d-mannose, d-galactose), a total sugar concentration of 54.8 g L−1 was achieved in the hydrolysate. The one-pot process combining hydrolytic enzyme production in solid-state fermentation with high solid loads during enzymatic hydrolysis of the milled SBPP reduces hydrolytic process costs by replacing chemical pre-treatments, enabling the in situ production of SBPP-adapted hydrolytic enzymes, as well as avoiding intermediate enzyme extraction and preparation steps. [ABSTRACT FROM AUTHOR]

Due to the high perishability of figs, calcium stands out as an important nutrient for orchard management. This study aims to study the pre-harvest applications of calcium chloride in fig orchards. The fig trees were sprayed with 0, 0.5, 1.0, 1.5 and 2.0% CaCl2, and fruits were thereafter stored for 0, 7, 14 and 21 d. The variables analysed were the concentration of calcium in leaves and fruits, weight loss, fruit decay, pectinolytic enzyme activity, and physical and chemical attributes of the fruits; CaCl2 sprays enhanced fruit Ca2+ concentration in leaves (14.03%) and fruits (29.3%) and were effective in reducing polygalacturonase and pectin methylesterase activity, weight loss and fruit decay. Pre-treatment with 1% CaCl2 provided fruits with larger diameters, greater firmness, and higher levels of total phenolic compounds in both fruit peel and pulp. The pre-harvest application at 1% CaCl2 showed to be a promising technique in producing high quality fruits and extended storage by approximately 7 to 10 days. These findings may be useful in planning new cultural practices for fig orchards that produce high-quality fruit with desirable characteristics for growers and consumers. [ABSTRACT FROM AUTHOR]

The deactivation of degrading and pectinolytic enzymes is crucial in the fruit juice industry. In commercial fruit juice production, a variety of approaches are applied to inactivate degradative enzymes. One of the most extensively utilized traditional procedures for improving the general acceptability of juice is thermal heat treatment. The utilization of a non-thermal pulsed electric field (PEF) as a promising technology for retaining the fresh-like qualities of juice by efficiently inactivating enzymes and bacteria will be discussed in this review. Induced structural alteration provides for energy savings, reduced raw material waste, and the development of new products. PEF alters the α-helix conformation and changes the active site of enzymes. Furthermore, PEF-treated juices restore enzymatic activity during storage due to either partial enzyme inactivation or the presence of PEF-resistant isozymes. The increase in activity sites caused by structural changes causes the enzymes to be hyperactivated. PEF pretreatments or their combination with other nonthermal techniques improve enzyme activation. For endogenous enzyme inactivation, a clean-label hurdle technology based on PEF and mild temperature could be utilized instead of harsh heat treatments. Furthermore, by substituting or combining conventional pasteurization with PEF technology for improved preservation of both fruit and vegetable juices, PEF technology has enormous economic potential. PEF treatment has advantages not only in terms of product quality but also in terms of manufacturing. Extending the shelf life simplifies production planning and broadens the product range significantly. Supermarkets can be served from the warehouse by increasing storage stability. As storage stability improves, set-up and cleaning durations decrease, and flexibility increases, with only minor product adjustments required throughout the manufacturing process. [ABSTRACT FROM AUTHOR]

Simple Summary: Grape skins, usually discarded during wine making, are a valuable source of cellulose (20–50%), hemicelluloses (15–20%), lignin (17–30%) and other compounds, e.g., polyphenols, which can be used as biomaterials in the manufacturing of a variety of new products, such as bioethanol or pharmaceutical products. However, to obtain these biomaterials, the complex polysaccharides of the grape cell walls must be broken down into smaller molecules to allow the extraction of compounds. The degradation process is often performed enzymatically or hydrothermally. Microorganisms that produce the required enzymes while using this waste product as a growth medium can have interesting economic advantages. Here, we created two genetically engineered wine yeast strains that produce grape cell wall degrading enzymes. These yeasts, when grown on grape pomace, induced enzymatic structural changes to the grape cell walls. A collection of antibodies binding to the different cell wall molecules were used to monitor the impact on the cell wall structure of the enzymes, confirming increased extractability of key cell wall polymers when relatively low levels of enzymes are present, illustrating the potential to develop and optimise yeast for grape waste valorisation applications. Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, β-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation. [ABSTRACT FROM AUTHOR]

Pectin is a major constituent of the plant cell wall, comprising compounds with important industrial applications such as homogalacturonan, rhamnogalacturonan and xylogalacturonan. A large array of enzymes is involved in the degradation of this amorphous substrate. The Glycoside Hydrolase 28 (GH28) family includes polygalacturonases (PG), rhamnogalacturonases (RG) and xylogalacturonases (XG) that share a structure of three to four pleated β-sheets that form a rod with the catalytic site amidst a long, narrow groove. Although these enzymes have been studied for many years, there has been no systematic analysis. We have collected a comprehensive set of GH28 encoding sequences to study their evolution in fungi, directed at obtaining a functional classification, as well as at the identification of substrate specificity as functional constraint. Computational tools such as Alphafold, Consurf and MEME were used to identify the subfamilies' characteristics. A hierarchic classification defines the major classes of endoPG, endoRG and endoXG as well as three exoPG classes. Ascomycete endoPGs are further classified in two subclasses whereas we identify four exoRG subclasses. Diversification towards exomode is explained by loops that appear inserted in a number of turns. Substrate-driven diversification can be identified by various specificity determining positions that appear to surround the binding groove. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting protein (PGIP) is an important plant biochemical anti-disease factor. PGIP has a leucine-rich repeat structure that can selectively bind and inhibit the activity of endo-polygalacturonase (endo-PG) in fungi, playing a key role in plant disease resistance. The regulation of PGIP in plant disease resistance has been well studied, and the effect of PGIP to increase disease resistance is clear. This review summarizes recent advances in understanding the PGIP protein structure, the PGIP mechanism of plant disease resistance, and anti-disease activity by PGIP gene transfer. This overview should contribute to a better understanding of PGIP function and can help guide resistance breeding of PGIP for anti-disease effects. [ABSTRACT FROM AUTHOR]

Pepper is one of the most important vegetable crops in China and has high economic value. However, the pepper fruit is easily softened and spoiled after harvest, which seriously affects its flavor, transportation, and economic value. In this study, we used pepper lines with different levels of storage resistance, A144 and A361, and performed physiological examination, transcriptomics, and metabolomics on them at 0 and 3 days after harvest in order to analyze their gene expression patterns and molecular regulatory mechanisms for storage tolerance. A total of 23,477 genes and 985 metabolites were identified. After comparing and analyzing each sample, we identified 7829 differentially expressed genes and 296 differential metabolites. We found that the genes such as ethylene-responsive transcriptional factor (ERFs), polygalacturonase (PG), cellulose synthase (CESA), abscisic acid insensitive (ABI), protein kinase 2 (SnRK2), and protein phosphatase 2C (PP2C) and metabolites such as phenylalanine and glycyl-tyrosine were differentially expressed between different storage times in the two materials. Through GO and KEGG enrichment analysis, we found that the differential genes were mainly enriched in carbohydrate metabolism, small molecule metabolism, and plant hormone signal transduction, and the differential metabolites were mainly enriched in flavonoid biosynthesis, glutathione metabolism, and cysteine and methionine metabolism pathways. This study provides a scientific basis for investigating the molecular mechanisms of storage tolerance and developing new pepper varieties with improved storage resistance. [ABSTRACT FROM AUTHOR]

Sheath blight disease of rice caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris) remains a global challenge due to the absence of reliable resistance genes and poor understanding of pathogen biology. Pectin, one of the most vital constituents of the plant cell wall, is targeted by pectin methylesterases, polygalacturonases, and few other enzymes of fungal pathogens. In this study, we catalogued the expressed genes of the fungal genome from RNAseq of R. solani infected four rice genotypes. Analysis of RNAseq revealed 3325 pathogen genes commonly expressed in all rice genotypes, in which 49, 490, and 83 genes were specific to BPT5204, Tetep, and Pankaj genotypes, respectively. To identify the early and late responding genes of R. solani during plant cell wall degradation, a real-time PCR analysis of 30 pectinolytic enzymes was done at six different time points after inoculation. The majority of these genes showed maximum induction at the 72 h time point, suggesting that it is the most crucial stage of infection. Pankaj showed lesser induction of these genes as compared to other genotypes. Leaf-blade tissue and 45 days old-growth stage are more favorable for the expression of pectin degradation genes of R. solani. Additionally, the expression analysis of these genes from four different strains of R. solani suggested differential regulation of genes but no distinct expression pattern between highly virulent and mild strains. The implications of the differential regulation of these genes in disease development have been discussed. This study provides the first such comprehensive analysis of R. solani genes encoding pectin degrading enzymes, which would help to decipher the pathogen biology and sheath blight disease development. [ABSTRACT FROM AUTHOR]

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations are a promising alternative to accelerate and optimize bioprocess development allowing for sophisticated cultivation experiments in high throughput. These include fed-batch and continuous cultivations with multiple options of process control and sample analysis which deliver valuable screening tools for industrial production. However, the model-based methods needed to operate these robotic facilities efficiently considering the complexity of biological processes are missing. We present an automated experiment facility that integrates online data handling, visualization and treatment using multivariate analysis approaches to design and operate dynamical experimental campaigns in up to 48 mini-bioreactors (8-12 mL) in parallel. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed, running and comparing 16 experimental conditions in triplicate. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable to different strains and cultivation strategies, and suitable for automatized process development reducing the experimental times and costs. [ABSTRACT FROM AUTHOR]

The polygalacturonase (PGU), hemicellulase (mannanase) and protease enzyme activities in commercial macerating, pectinase-enzyme preparations commonly used by wineries in Ontario (Scottzyme Color X and Color Pro) were measured under various simulated process conditions (temperature, pH, and ethanol concentration). Treatments included three temperatures (15, 20 and 30 °C; pH = 3.0, 3.5, 4.0 and 5.0; ethanol = 0%), four pH levels (3.0, 3.5, 4.0 and 5.0; temperature = 15, 20, 30 and 50 °C; ethanol = 0%), and four ethanol concentrations ((2.5, 5, 7.5 and 10%); temperature = 20 °C and pH = 3.5.) Polygalacturonase enzyme activity in Color X increased linearly with temperature at all pH levels, and increased with pH at all temperature regimes. Polygalacturonase activity decreased with increasing ethanol. Color X mannanase activity increased with temperatures between 15 and 40 °C, and decreased with increased pH between 3.0 and 5.0. Response of mannanase to ethanol was cubic with a sharp decrease between 8 and 10% ethanol. Protease activity increased linearly with temperatures between 20 and 40 °C. These data suggest that the PGU, mannanase and protease components in these enzyme products provide sufficient activities within the ranges of pH, temperature, and ethanol common during the initial stages of red wine fermentations, although low must temperatures (<20 °C) and presence of ethanol would likely lead to sub-optimal enzyme activities. [ABSTRACT FROM AUTHOR]

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 "QinWang" (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. [ABSTRACT FROM AUTHOR]

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose) and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution. [ABSTRACT FROM AUTHOR]

Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL-1 and 101.01 Units·mg·protein-1·min-1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3-7 and >50% activity in 10-50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris. [ABSTRACT FROM AUTHOR]

Peeled and unpeeled celeriac pulp was macerated with pectinase from Rhizopus sp. at 25 °C for 30 and 60 min. Peeling, enzyme addition, and maceration time significantly affected the quality characteristics of the juice. The juice obtained from peeled celeriac was characterized by higher pressing yield, sucrose content, and antioxidant activity (ABTS *+ and DPPH * ). The juice from the unpeeled root had higher extract, fructose, glucose, total polyphenols, antioxidant activity (FRAP), total phenolic acids, and total luteolin content. Applying the enzyme to celeriac pulp had no significant effect on the extract's content, analyzed sugars, and antioxidant activity of the juices (ABTS *+ ). Adding pectinase to unpeeled celery pulp resulted in a 2-10% increase in pressing efficiency, compared to the control sample held at 25 °C for the same period. Maceration of the enzyme-peeled pulp increased the antioxidant potential of the juice by 22% in the FRAP method. In contrast, in all juices analyzed, unpeeled and peeled roots increased antioxidant activity measured by the DPPH* method by 24-57% and total phenolic acids by 20-57%. The time of holding the pulp at 25 °C was an important factor, and its extension resulted in a decrease in the values of most of the analyzed parameters, with the exception of pressing efficiency and fructose content in all analyzed juice samples. Short-term, 30-min maceration of peeled and unpeeled celery pulp with pectinase from Rhizopus sp. had a significant effect on increasing juice yield, antioxidant activity, and phenolic compound content.

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg-1 and 0.31 mg·mL-1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%-34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry. [ABSTRACT FROM AUTHOR]

D-limonene (4-isopropenyl-1-methylcyclohexene) is an important compound in several citrus essential oils (such as orange, lemon, tangerine, lime, and grapefruit). It has been used as a flavoring agent and as a food preservative agent, with generally recognized as safe (GRAS) status. D-limonene has been well-studied for its anti-inflammatory, antioxidant, anti-cancer, and antibacterial properties. The antibacterial activity of D-limonene against food-borne pathogens was investigated in this study by preparing a D-limonene nanoemulsion. The D-limonene solution and nanoemulsion have been prepared in six concentrations, 0.04%, 0.08%, 0.1%, 0.2%, 0.4%, and 0.8% ( v / v ), respectively, and the antibacterial activity was tested against four food-borne pathogens ( Staphylococcus aureus , Listeria monocytogenes , Salmonella enterica , and Escherichia coli ). The results showed that the D-limonene nanoemulsion had good nanoscale and overall particle size uniformity, and its particle size was about 3~5 nm. It has been found that the D-limonene solution and nanoemulsion have a minimal inhibitory concentration of 0.336 mg/mL, and that they could inhibit the growth of microorganisms efficiently. The data indicate that the D-limonene nanoemulsion has more antibacterial ability against microorganisms than the D-limonene essential oil. After bananas are treated with 1.0% and 1.5% D-limonene nanoemulsion coatings, the water loss of the bananas during storage and the percentage of weight loss are reduced, which can inhibit the activity of pectinase. The application of a biocoating provides a good degree of antibacterial activity and air and moisture barrier properties, which help with extending the shelf life of bananas.

The metabolism of polysaccharides in the Zizania latifolia cell wall helps maintain the postharvest quality during storage. Fresh Z. latifolia was stored at 4 °C and 25 °C to evaluate the hardness, cell wall polysaccharide composition, cell wall structure, active ingredients, and cell wall metabolism-related enzyme activities. The results showed that hardness declined concomitantly with an increase in water-soluble pectin content during storage, as well as with a decrease in propectin and cellulose contents. Correlation analysis showed that lower activities of cell wall-degrading enzymes, such as polygalacturonase, cellulase, and β-galactosidase in Z. latifolia stored at 4 °C, were associated with lighter fiberization and greater hardness, compared with those stored at 25 °C. Additionally, the results of infrared spectroscopy showed that texture softening may be attributed to a decrease in the degree of esterification of water-soluble polysaccharides at 25 °C compared to that at 4 °C. [ABSTRACT FROM AUTHOR]

Polygalacturonases (PG) are pectinolytic enzymes that have technological, functional and biological applications in food processing, fruit ripening and plant-fungus interactions, respectively. In the present, a microtitre plate methodology was used for rapid screening of 61 isolates of fungi from Aspergillus section Nigri to assess production of endo- and exo-PG. Studies of scale-up were carried out in a fixed bed reactor operated under different parameters using the best producer strain immobilised in orange peels. Four experiments were conducted under the following conditions: the immobilised cells without aeration; immobilised cells with aeration; immobilised cells with aeration and added pectin; and free cells with aeration. The fermentation was performed for 168 h with removal of sample every 24 h. Aspergillus niger strain URM 5162 showed the highest PG production. The results obtained indicated that the maximum endo- and exo-PG activities (1.18 U·mL-1 and 4.11 U·mL-1, respectively) were obtained when the reactor was operating without aeration. The microtitre plate method is a simple way to screen fungal isolates for PG activity detection. The fixed bed reactor with orange peel support and using A. niger URM 5162 is a promising process for PG production at the industrial level. [ABSTRACT FROM AUTHOR]

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12-16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts., Competing Interests: The authors declare no conflict of interest.

Landoltia punctata, a widely distributed duckweed strain with the ability to accumulate starch, was used as a novel feedstock for bioethanol production by Saccharomyces cerevisiae. To improve ethanol production, pectinase pretreatment was used to release much more glucose from L. punctata mash and the pretreatment conditions (enzyme loading, temperature and pretreatment time) for the duckweed were optimized by using a surface response design. The results showed that maximum glucose yield was 218.64 ± 3.10 mg/g dry matter, which is a 142% increase compared to the untreated mash, with a pectinase dose of 26.54 pectin transeliminase unit/g mash at 45 °C for 300 min. Pectinase pretreatment apparently changed the ultrastructure of L. punctata, as evidenced by scanning electron microscopy analysis. Further fermentation experiments were performed and 30.8 ± 0.8 g/L of ethanol concentration, 90.04% of fermentation efficiency and 2.20 g/L/h of productivity rate were achieved. This is the highest ethanol concentration reported to date using duckweed as the feedstock. [ABSTRACT FROM AUTHOR]

Response surface methodology (RSM) along with central composite design (CCD) was applied to optimize the freeze drying conditions for purified pectinase from mango (Mangifera indica cv. Chokanan) peel. The effect of pectinase content (-2.66, 62.66 mg/mL), Arabic gum (-1.21, 10.21%, w/v), and maltodextrin (0.73, 7.26%, w/v) as independent variables on activity, yield, and storage stability of freeze-dried enzyme was evaluated. Storage stability of pectinase was investigated after one week at 4 °C and yield percentage of the enzyme after encapsulation was also determined. The independent variables had the most significant (p < 0.05) effect on pectinase activity and yield of the enzyme. It was observed that the interaction effect of Arabic gum and maltodextrin improved the enzymatic properties of freeze-dried pectinase. The optimal conditions for freeze-dried pectinase from mango peel were obtained using 30 mg/mL of pectinase content, 4.5 (%, w/v) of Arabic gum, and 4 (%, w/v) of maltodextrin. Under these conditions, the maximum activity (11.12 U/mL), yield (86.4%) and storage stability (84.2%) of encapsulated pectinase were achieved. [ABSTRACT FROM AUTHOR]

Ganoderma lucidum and Ganoderma sinense, known as Lingzhi in Chinese, are commonly used Chinese medicines with excellent beneficial health effects. Triterpenes and polysaccharides are usually considered as their main active components. However, the content of triterpenes differs significantly between the two species of Ganoderma. To date, a careful comparison of polysaccharides from the two species of Ganoderma has not been performed. In this study, polysaccharides from fruiting bodies of two species of Lingzhi collected from different regions of China were analyzed and compared based on HPSEC-ELSD and HPSEC-MALLS-RI analyses, as well as enzymatic digestion and HPTLC of acid hydrolysates. The results indicated that both the HPSEC-ELSD profiles and the molecular weights of the polysaccharides were similar. Enzymatic digestion showed that polyshaccharides from all samples of Lingzhi could be hydrolyzed by pectinase and dextranase. HPTLC profiles of their TFA hydrolysates colored with different reagents and their monosaccharides composition were also similar. [ABSTRACT FROM AUTHOR]

An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel. [ABSTRACT FROM AUTHOR]

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0–26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides. [ABSTRACT FROM AUTHOR]

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes. [ABSTRACT FROM AUTHOR]

Fruit cracking is a physiological disease that occurs during fruit development, which limits the quality and marketability of the fruit and causes great economic losses. Fruit cracking is affected by physiological, genetic and environmental factors. In this paper, the mechanism of fruit cracking was elaborated from cutin and cell wall, especially the gene families related to cell wall metabolism, including the polygalacturonase (PG) gene family, xylologlucan endotransglucosylase/hydrolase (XTH) gene family and expansin gene family. In addition, due to the advancement of high-throughput sequencing technology, an increasing number of horticultural plants have completed genome sequencing. This paper expounds the application of omics, including transcriptome, proteome, metabolomics and integrative omics in fruit cracking. The measures to reduce fruit cracking include using plastic rain covers and bagging, and spraying mineral and plant growth regulators. In this paper, the mechanisms of fruit cracking are reviewed at the molecular level, and the problems needing to be solved in fruit cracking research are put forward. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is a hemibiotrophic pathogen responsible for great losses in dicot crop production. An ExoPG gene (VDAG_03463,) identified using subtractive hybridization/cDNA-AFLP, showed higher expression levels in highly aggressive than in weakly aggressive V. dahliae isolates. We used a vector-free split-marker recombination method with PEG-mediated protoplast to delete the ExoPG gene in V. dahliae. This is the first instance of using this method for V. dahliae transformation. Only two PCR steps and one transformation step were required, markedly reducing the necessary time for gene deletion. Six mutants were identified. ExoPG expressed more in the highly aggressive than in the weakly aggressive isolate in response to potato leaf and stem extracts. Its expression increased in both isolates during infection, with higher levels in the highly aggressive isolate at early infection stages. The disruption of ExoPG did not influence virulence, nor did it affect total exopolygalacturonase activity in V. dahliae. Full genome analysis showed 8 more genes related to polygalacturonase/pectinase activity in V. dahliae. Transcripts of PGA increased in the △exopg mutant in response to potato leaf extracts, compared to the wild type. The expression pattern of those eight genes showed similar trends in the △exopg mutant and in the weakly aggressive isolate in response to potato extracts, but without the increase of PGA in the weakly aggressive isolate to leaf extracts. This indicated that the △exopg mutant of V. dahliae compensated by the suppression of ExoPG by activating other related gene. [ABSTRACT FROM AUTHOR]

Scouring is one of the initial steps in the processing of natural textile fibers (e.g., cotton), performed to remove waxes and pectins, together with spinning oils and other impurities of the plant cell cuticle. Traditional chemical bleaching with boiling NaOH led to harsh removal of the entire fabric's cuticle waxy layer accompanied by an unwanted alkaline waste. Extracellular lytic enzymes such as lipases, cellulases and pectinases play an essential role in host plant-pathogen interactions. They degrade the plant cuticle and tissue and enable pathogen invasion. Such enzymes, specifically cutinase and pectinase, have been considered potential bio-scouring agents to degrade the cotton fabric cuticle's outer layer at low temperature and alleviate environmental pollution. In this work, the combined effect of cutinase, pectin lyase, or polygalacturonase on the scouring of cotton fabrics was studied using evaporative light-scattering reverse-phase HPLC and GC-MS analysis of the reaction components, and measuring changes in the cotton fabrics' properties. The traditional method of cotton fabrics' scouring with NaOH resulted in decreased pectin content and increased cellulose fibers accessibility, evaluated by specific staining. Treating the cotton fibers' cuticle with cutinase led to the acidification of the reaction mixture, a decrease in enzyme-specific activity, and elevation in hexadecanoic acid and octadecanoic acids in the reaction fluid. These two saturated fatty acids are the main wax constituents of raw cotton fabrics, identified using GC-MS after dichloromethane reflux overnight. Treating cotton fabrics with each of the three enzymes, cutinase, pectin lyase, or polygalacturonase, increased their pectin removal, as measured by high concentrations of D-galacturonic acid and other pectin constituents in the reaction fluid. A synergistic effect was found in the combined treatment of cutinase and pectin lyase in the hydrolysis of the cotton fibers' cuticle. This effect was expressed in high water absorbency of the treated fibers, increased fabric weight loss and sharp elevation of a cutin and pectin monomer's related peaks (retention time [RT] = 4.1 min and 2.9, 4.5 min, respectively). A model was suggested for the synergistic action between cutinase and pectin lyase. It assumes that the cuticle's digestion by cutinase results in the enlargement and formation of outer layer micropores, which enables the rapid penetration of pectinase into the inner pectin layer. [ABSTRACT FROM AUTHOR]

Fragments of wood drifting in the vicinity of Spitzbergen were used for the isolation of microorganisms, carried out using atypical carbon sources: colloidal chitin, cellulose and carboxymethylcellulose, xylan, casein, tributrin and olive oil. Purified cultures were subjected to a three-step identification: with classical methods, using MALDI-TOF MS Biotyper whole-cell protein fingerprinting, and molecular analysis of 16S rDNA. Subsequently, a preliminary assessment of the enzymatic potential of isolates was carried out. As a result, cellulolytic activity was observed in more than 50% of the bacterial strains, exhibiting activity of 0.30–0.40 U/mL. Over 53% of the isolates demonstrated xylanolytic activity, of which the highest reached from 0.40 to 0.90 U. Polygalacturonase activity of 0.003–1.6 was also demonstrated in half of the bacterial strains studied. Proteolytic activity of isolates did not exceed 0.3 U. An important highlight was the ability of fluorescent dye production by certain strains, grown on skim milk-agar, but also on pure meat extract. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the "Guangfen-1" and "Baxi" varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polygalacturonase (PG) is an essential hydrolytic enzyme responsible for pectin degradation and thus plays an important role in fruit softening and other cell separation processes. PG protein is encoded by a multigene family, however, the members of PG gene family in kiwifruit (Actinidia chinensis) have not been extensively identified. In this study, a total of 51 AcPG genes in kiwifruit genome were identified. They are phylogenetically clustered into seven clades, and of them AcPG4 and AcPG18 with other known PG genes involved in fruit softening from peach, pear, papaya and melon form a small cluster together. The members of kiwifruit PG gene family consist of three to nine exons and two to eight introns, and their exon/intron structures are generally conserved in all clades except the clade D and E. During fruit softening of kiwifruit 'Donghong' under ambient temperature, cell wall modifying enzymes, including PG, PL (pectate and pectin lyases), and PE (pectinesterase, also known as pectin methylesterase, PME) showed a different activity profile, and of them, PG and PE activities largely correlated with the change of pectin content and firmness. Moreover, only 11 AcPG genes were highly or moderately expressed in softening fruit, and of which three AcPG genes (AcPG4, AcPG18, and AcPG8, especially the former two) has been found to strongly correlate with the profile of PG activity and pectin content, as well as fruit firmness, suggesting that they maybe play an important role in fruit softening. Thus, our findings not only benefit the functional characterization of kiwifruit PG genes, but also provide a subset of potential PG candidate genes for further genetic manipulation. [ABSTRACT FROM AUTHOR]