Enzyme Activity Analysis Research Articles

Analysis of seed production and seed shattering in a new artificial grassland forage: pigeon pea.

Pigeon pea is a perennial leguminous plant that is widely cultivated as a forage and pharmaceutical plant in subtropical and tropical areas, especially in artificial grasslands. Higher seed shattering is one of the most important factors in potentially increasing the seed yield of pigeon pea. Advance technology is necessary to increase the seed yield of pigeon pea. Through 2 consecutive years of field observations, we found that fertile tiller number was the key component of the seed yield of pigeon pea due to the direct effect of fertile tiller number per plant (0.364) on pigeon pea seed yield was the highest. Multiplex morphology, histology, and cytological and hydrolytic enzyme activity analysis showed that shatter-susceptible and shatter-resistant pigeon peas possessed an abscission layer at the same time (10 DAF); however, abscission layer cells dissolved earlier in shattering-susceptible pigeon pea (15 DAF), which led to the tearing of the abscission layer. The number of vascular bundle cells and vascular bundle area were the most significant negative factors (p< 0.01) affecting seed shattering. Cellulase and polygalacturonase were involved in the dehiscence process. In addition, we inferred that larger vascular bundle tissues and cells in the ventral suture of seed pods could effectively resist the dehiscence pressure of the abscission layer. This study provides foundation for further molecular studies to increase the seed yield of pigeon pea.

Antimicrobial mechanisms of ZnO nanoparticles to phytopathogen Pseudomonas syringae: Damage of cell envelope, suppression of metabolism, biofilm and motility, and stimulation of stomatal immunity on host plant

Nanoparticles have recently been employed as a new strategy to act as bactericides in agricultural applications. However, the effects and mechanisms of foliar deposition of nanoparticles on bacterial pathogens, plant physiology and particularly plant immunity have not been sufficiently understood. Here, we investigated the effects and mechanisms of ZnO NPs in controlling of tobacco wildfire caused by Pseudomonas syringae pv. tabaci, through the comprehensive analysis of biological changes of both bacteria and plants. The global gene expression changes of Pseudomonas syringae pv. tabaci supported that the functions of “protein secretion”, “membrane part”, “signal transducer activity”, “locomotion”, “chemotaxis” and “taxis” in bacteria, as well as the metabolic pathways of “bacterial chemotaxis”, “two-component system”, “biofilm formation”, “ABC transporters” and “valine, leucine and isoleucine degradation” were significantly down-regulated by ZnO NPs. Correspondingly, we reconfirmed that the cell envelope structure, biofilm and motility of Pseudomonas syringae pv. tabaci were directly disrupted or suppressed by ZnO NPs. Different from completely killing Pseudomonas syringae pv. tabaci, ZnO NPs (0.5 mg/mL) potentially improved plant growth and immunity through enzymatic activity and global molecular response analysis. Furthermore, the changes of gene expression in ABA signaling pathway, ABA concentration and stomatal aperture all supported that ZnO NPs can specifically stimulate stomatal immunity, which is important to defend bacterial infection. Taken together, we proposed that both the inhibition or damage of motility, biofilm, metabolisms, virulence and cell envelope on P. syringae pv. tabaci, and the activation of the stomatal immunity formed two-layered antibacterial mechanisms of ZnO NPs on phytopathogenic bacteria.

Fe-loaded alginate hydrogel beads activating peroxymonosulfate for enhancing anaerobic fermentation of waste activated sludge: Performance and potential mechanism

The recovery of volatile fatty acids (VFAs) through anaerobic fermentation (AF) is usually restricted by the poor biodegradability of waste activated sludge (WAS). This study proposed a novel strategy, i.e. peroxymonosulfate (PMS) activated by Fe-loaded sodium alginate hydrogel beads (Fe-SA), to enhance AF performance. Experimental results demonstrated that the as-synthesized Fe-SA and PMS co-pretreatment synergistically enhanced WAS solubilization and VFAs production. The maximal VFAs yield of 2013 mg COD/L was achieved at the Fe-SA dosage of 4.0 mM/g TSS, which was 93.7% higher than that with sole PMS addition and 8.82 times higher than that of the control. Mechanistic studies elucidated that the generation of reactive radicals such as SO4•− and •OH from PMS was greatly induced by Fe-SA, which contributed to WAS disintegration and degradation of refractory compounds. Additionally, analysis of the key enzyme activities indicated that the Fe-SA could strengthen biological hydrolysis and acidogenesis of sludge during AF. Microbial analysis illustrated that Fe-SA evidently improved the abundances of fermentative microorganisms as well as functional gene expression via creating a favorable environment for microbial growth. This study demonstrated the applicable potential of Fe-SA hydrogel beads activating PMS for VFAs production and provides an important reference for developing advanced oxidation processes-based application in AF.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Inhibition of GSK3B phosphorylation improves glucose and lipid metabolism disorder

Hepatic glycolipid metabolism disorder is considered as one of the key pathogenic factors for many chronic diseases. Revealing the molecular mechanism of metabolic disorder and exploring drug targets are crucial for the treatment of glucose and lipid metabolic diseases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to be associated with the pathogenesis of various metabolic diseases. Herein, GAPDH-knockdown ZFL cells and GAPDH-downregulation zebrafish exhibited significant lipid deposition increase and glycogen reduction, thus inducing glucose and lipid metabolism disorders. Using high-sensitivity mass spectrometry-based proteomic and phosphoproteomic analysis, we identified 6838 proteins and 3738 phosphorylated proteins in GAPDH-knockdown ZFL cells. The protein-protein interaction network and DEPPs analyses showed that gsk3baY216 were involved in lipid and glucose metabolism, which was verified by In vitro study. The enzyme activity analysis and cell staining results showed that HepG2 and NCTC-1469 cells transfected with GSK3BY216F plasmid had significantly lower glucose and insulin levels, the decreased lipid deposition, and the increased glycogen synthesis than those transfected with GSK3BY216E plasmid, suggesting that inhibition of GSK3B phosphorylation could significantly improve GSK3B hyperphosphorylation-induced glucose tolerance impairment and insulin sensitivity reduction. To our knowledge, this is the first multi-omic study of GAPDH-knockdown ZFL cells. This study provides insights into the molecular mechanism of glucose and lipid metabolic disorder, and provides potential targets (kinases) for the treatments of human glucose and lipid metabolic diseases.

Effects of adaptation diet and exogenous enzymes on true metabolizable energy and cecal microbial ecology, short-chain fatty acid profile, and enzyme activity in roosters fed barley and rye diets

Three experiments evaluated effects of adaptation diet and exogenous β-glucanase and xylanase on TMEn of barley and rye. Single Comb White Leghorn roosters were fed adaptation diets based on corn/soybean meal (SBM), barley/SBM with and without β-glucanase, or rye/corn/SBM with and without xylanase for 4 wk. In Experiments 1 and 2, after the adaptation period, TMEn was determined using a 48 h precision-fed rooster assay for 100% barley or 100% rye diets with or without β-glucanase or xylanase, respectively. Experiment 3 consisted only of feeding adaptation diets for 4 wk. Cecal samples were collected at the end of experiments for microbial ecology, short-chain fatty acid (SCFA) profiles, and enzyme activity analyses. In Experiments 1 and 2, β-glucanase increased (P < 0.05) TMEn of barley, and there was no significant effect of adaptation diet on TMEn values. Total cecal Eubacteria and Ruminococcaceae were decreased (P < 0.05) and Escherichia coli were increased (P < 0.05) at the end of the TMEn assay compared with the end of the adaptation period (with no TMEn assay). There was a large decrease (P < 0.05) for most cecal SCFA at the end of the TMEn assay compared with the end of the adaptation period. Both cecal β-glucanase and xylanase activity were increased for birds fed adaptation diets containing the respective enzyme. In Experiment 3, there were no consistent effects of adaptation diet on cecal microbial profiles or SCFA but cecal β-glucanase activity was increased (P < 0.05) by exogenous β-glucanase for barley and cecal xylanase activity was increased (P < 0.05) by exogenous xylanase for rye. Overall, the results indicated that TMEn of barley was increased by exogenous β-glucanase, adaptation diet did not significantly influence the TMEn response to the dietary enzymes, and cecal fermentation (based on cecal SCFA) was greatly reduced by the TMEn assay. Cecal β-glucanase and xylanase activity, however, were often increased by feeding high barley and high rye diets containing exogenous enzymes.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Mechanisms for the structural dependent transformation of 6:2 and 8:2 polyfluoroalkyl phosphate diesters in wheat (Triticum aestivum L.)

Polyfluoroalkyl phosphate esters (PAPs) are widely used and detected in various environmental media and organisms, but little is known about their behaviors in plants. In this study, the uptake, translocation and transformation of 6:2 and 8:2 diPAP in wheat using hydroponic experiments were investigated. 6:2 diPAP was more easily taken up by roots and translocated to shoots than 8:2 diPAP. Their phase I metabolites were fluorotelomer saturated carboxylates (FTCAs), fluorotelomer unsaturated carboxylates (FTUCAs) and perfluoroalkyl carboxylic acids (PFCAs). PFCAs with even-numbered chain length were the primary phase I terminal metabolites suggesting that they were mainly generated through β-oxidation. Cysteine and sulfate conjugates were the primary phase II transformation metabolites. The higher levels and ratios of phase II metabolites in the 6:2 diPAP exposure group indicated that the phase I metabolites of 6:2 diPAP were more susceptible to phase II transformation than that of 8:2 diPAP, which was confirmed by density functional theory calculation. Enzyme activity analyses and in vitro experiments demonstrated that cytochrome P450 and alcohol dehydrogenase actively participated in the phase Ⅰ transformation of diPAPs. Gene expression analyses showed that glutathione S-transferase (GST) was involved in the phase Ⅱ transformation, and the subfamily GSTU2 played a dominant role.

Reactive Oxygen Species Metabolism Modulation on the Quality of Apple Fruits Inoculated with Penicillium expansum under Different Ambient pHs

Apple blue mold is a significant postharvest disease caused by Penicillium expansum. pH modification in colonized tissues leads to the production of organic substances, the modulation of enzymes, and then increases fungal pathogenicity. This study evaluated Penicillium expansum-inoculated apple fruits’ quality responding to pH treatments ranging from 2.5 to 8.5 and analyzed the reactive oxygen species (ROS) metabolism modulation in inoculated apple fruits at the same pH. The results showed that the fruit quality of the firmness, total soluble solids, and titratable acid displayed a quick loss at pHs 5.0 and 7.0, compared with 2.5 and 8.5. Similarly, higher disease incidence was observed at pHs 5.0 and 7.0. Apple fruits infected with P. expansum at pHs 2.5 and 8.5 had less content of O2•−, H2O2, and malondialdehyde (MDA); lower enzymatic activity of NADPH oxidase (NOX); and greater cell membrane integrity than those at pHs 5.0 and 7.0. The analysis of the antioxidant enzymatic activities showed upregulation of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) at pHs 2.5 and 8.5 compared with those at pHs 5.0 and 7.0. Similar trends were shown in ascorbic acid and glutathione. These results support the hypothesis that inoculated apple fruits at pHs 2.5 and 8.5 improve resistance to P. expansum by modulating ROS metabolism, compared with pHs 5.0 and 7.0.

Kinetic and Regulatory Properties of Yarrowia lipolytica Aconitate Hydratase as a Model-Indicator of Cell Redox State under pH Stress.

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction.

Screening and Identification of Potato StSPS1, a Potential Crucial Gene Regulating Seed Potato Vigor

Sucrose phosphate synthase (SPS), a crucial rate-limiting enzyme that catalyzes the synthesis of precursors of sucrose, plays an indispensable role in the regulation of seed potato vigor. Nonetheless, the genes that encode SPS in potato have not undergone complete analysis, and the primary genes responsible for the regulation of seed potato vigor have not been screened and identified. In this study, four StSPS family members were identified by comparing the potato genome database with homologous proteins. Using bioinformatics, the physicochemical properties, subcellular localization, sequence structure, conserved motifs, and phylogenetics of StSPS were analyzed. The expression levels of StSPS in different potato tissues, from dormancy to sprouting in the seed potato tubers, were measured via qRT-PCR. The expression data from the potato genome database and previous transcriptome and proteome studies of dormancy to sprouting were also compared. After combining the analysis of SPS enzyme activity in diverse tuber tissues and the correlation analysis between multiple varieties with different dormancy periods and the expression of StSPS1, the primary gene StSPS1 that might regulate seed potato vigor was identified. This study set a theoretical and experimental groundwork for further verification and clarification of the regulatory function of StSPS1 in alterations in seed potato vigor.

DNA accelerates the protease inhibition of a bacterial serpin chloropin.

Serine protease inhibitors (Serpins) are the most widely distributed protease inhibitors in nature and have been identified from all kingdoms of life. Eukaryotic serpins are most abundant with their activities often subject to modulation by cofactors; however, little is known about the regulation of prokaryotic serpins. To address this, here we prepared a recombinant bacteria serpin, termed chloropin, derived from green sulfur bacteria Chlorobium limicola and solved its crystal structure at 2.2Å resolution. This showed a canonical inhibitory serpin conformation of native chloropin with a surface-exposed reactive loop and a large central beta-sheet. Enzyme activity analysis showed that chloropin could inhibit multiple proteases, such as thrombin and KLK7 with second order inhibition rate constants at 2.5×104M-1s-1 and 4.5×104M-1s-1 respectively, consistent with its P1 arginine residue. Heparin could accelerate the thrombin inhibition by ∼17-fold with a bell-shaped dose-dependent curve as seen with heparin-mediated thrombin inhibition by antithrombin. Interestingly, supercoiled DNA could accelerate the inhibition of thrombin by chloropin by 74-fold, while linear DNA accelerated the reaction by 142-fold through a heparin-like template mechanism. In contrast, DNA did not affect the inhibition of thrombin by antithrombin. These results indicate that DNA is likely a natural modulator of chloropin protecting the cell from endogenous or exogenous environmental proteases, and prokaryotic serpins have diverged during evolution to use different surface subsites for activity modulation.

Multiparametric analysis of proteolytic enzyme activity using mass cytometry

Enzymy proteolityczne, inaczej proteazy, są katalizatorami komórkowymi, które odpowiedzialne są za przyspieszenie reakcji hydrolizy wiązania amidowego w substratach białkowych. Proteazy stanowią kluczowy element w wielu ścieżkach sygnalizacyjnych komórki oraz umożliwiają utrzymanie homeostazy. Aktywność tych enzymów jest kontrolowana przez wewnątrzkomórkowe inhibitory, które uniemożliwiają nadmierną hydrolizę substratów białkowych. W przypadku mutacji genowych bądź chorób prowadzących do zaburzenia działania enzymów proteolitycznych, istnieje możliwość wystąpienia poważnych stanów patofizjologicznych, takich jak m.in. choroby neurodegeneracyjne, nowotworzenie czy zaburzenia krzepnięcia krwi. W związku z powyższym, precyzyjna analiza aktywności poszczególnych proteaz jest niezwykle istotna w ocenie stanu fizjologicznego poszczególnych komórek czy tkanek. Do dziś złotym standardem w tego typu badaniach jest zastosowanie tzw. markerów chemicznych, które nieodwracalnie łączą się z celowanym białkiem bądź grupą białek, tylko i wyłącznie w ich katalitycznie aktywnej formie, umożliwiając ich detekcję w próbkach biologicznych. Głównym problemem w zastosowaniu tych narzędzi chemicznych jest fakt, iż wiele proteaz współpracuje ze sobą w niezwykle złożonych komórkowych sieciach sygnalizacyjnych. W związku z tym, niezbędne jest przeprowadzenie wieloparametrycznej analizy, która ukaże wszystkie badane elementy aktywnie działające w obrębie danego systemu. W niniejszej pracy opisano główne założenia rozprawy doktorskiej dr inż. Katarzyny Groborz, które odnoszą się do projektowania, syntezy oraz walidacji nowego typu narzędzi chemicznych do badania aktywności proteaz przy użyciu cytometrii masowej. Otrzymane narzędzia, nazwane markerami chemicznymi typu TOF (ang. Time Of Flight), posiadają zalety tradycyjnych markerów chemicznych oraz umożliwiają wieloparametryczną analizę złożonych systemów biologicznych przy użyciu nowoczesnej technologii, jaką jest cytometria masowa.

Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Distinct Changes in Placental Ceramide Metabolism Characterize Type 1 and 2 Diabetic Pregnancies with Fetal Macrosomia or Preeclampsia.

Disturbances of lipid metabolism are typical in diabetes. Our objective was to characterize and compare placental sphingolipid metabolism in type 1 (T1D) and 2 (T2D) diabetic pregnancies and in non-diabetic controls. Placental samples from T1D, T2D, and control pregnancies were processed for sphingolipid analysis using tandem mass spectrometry. Western blotting, enzyme activity, and immunofluorescence analyses were used to study sphingolipid regulatory enzymes. Placental ceramide levels were lower in T1D and T2D compared to controls, which was associated with an upregulation of the ceramide degrading enzyme acid ceramidase (ASAH1). Increased placental ceramide content was found in T1D complicated by preeclampsia. Similarly, elevated ceramides were observed in T1D and T2D pregnancies with poor glycemic control. The protein levels and activity of sphingosine kinases (SPHK) that produce sphingoid-1-phosphates (S1P) were highest in T2D. Furthermore, SPHK levels were upregulated in T1D and T2D pregnancies with fetal macrosomia. In vitro experiments using trophoblastic JEG3 cells demonstrated increased SPHK expression and activity following glucose and insulin treatments. Specific changes in the placental sphingolipidome characterize T1D and T2D placentae depending on the type of diabetes and feto-maternal complications. Increased exposure to insulin and glucose is a plausible contributor to the upregulation of the SPHK-S1P-axis in diabetic placentae.

APPLICATION OF Azospirillum brasilense AND NITROGEN IN TOPDRESSING IN CORN HYBRIDS

Corn (Zea mays L.) is the most cultivated cereal in the world. The high yields obtained for this crop are mainly linked to nitrogen requirements. Technologies that aim to reduce costs with nitrogen fertilizers, with less environmental impact, such as diazotrophic bacteria, should be considered an advantageous alternative for this crop. The present study aimed to evaluate two corn hybrids, BioGene 7046® and Dekalb 310®, with three topdressing nitrogen doses (0.35 and 70 kg ha-1 of N) associated with four application methods of Azospirillum brasilense (without inoculation, seed inoculation, foliar inoculation, and the combination of inoculation via seeds and leaves). A randomized block design with four replications arranged in a 2x3x4 factorial scheme was used to evaluate variables related to growth and development, physiological analysis of nitrate reductase enzyme activity, and yield variables and their components. The use of Azospirillum brasilense did not affect the development of corn plants and did not increase grain yield. In the edaphoclimatic conditions in which this research was developed, nitrogen fertilization at doses of 35 and 70 kg ha-1 benefit nutrition, development, and corn yield components but are insufficient to increase grain yield.

Soil arthropods promote litter enzyme activity by regulating microbial carbon limitation and ecoenzymatic stoichiometry in a subalpine forest

Soil arthropods are crucial decomposers of litter at both global and local scales, yet their functional roles in mediating microbial activity during litter decomposition remain poorly understood. Here, we conducted a two-year field experiment using litterbags to assess the effects of soil arthropods on the extracellular enzyme activities (EEAs) in two litter substrates (Abies faxoniana and Betula albosinensis) in a subalpine forest. A biocide (naphthalene) was used to permit (nonnaphthalene) or exclude (naphthalene application) the presence of soil arthropods in litterbags during decomposition. Our results showed that biocide application was effective in reducing the abundance of soil arthropods in litterbags, with the density and species richness of soil arthropods decreasing by 64.18–75.45 % and 39.19–63.30 %, respectively. Litter with soil arthropods had a greater activity of C-degrading (β-glucosidase, cellobiohydrolase, polyphenol oxidase, peroxidase), N-degrading (N-acetyl-β-D-glucosaminidase, leucine arylamidase) and P-degrading (phosphatase) enzymes than litter from which soil arthropods were excluded. The contributions of soil arthropods to C-, N- and P-degrading EEAs in the fir litter were 38.09 %, 15.62 % and 61.69 %, and those for the birch litter were 27.97 %, 29.18 % and 30.40 %, respectively. Furthermore, the stoichiometric analyses of enzyme activity indicated that there was potential C and P colimitation in both the soil arthropod inclusion and exclusion litterbags, and the presence of soil arthropods decreased C limitation in the two litter species. Our structural equation models suggested that soil arthropods indirectly promoted C-, N- and P-degrading EEAs by regulating the litter C content and litter stoichiometry (e.g., N/P, LN/N and C/P) during litter decomposition. These results demonstrate that soil arthropods play an important functional role in modulating EEAs during litter decomposition.

Targeted multi-platform metabolome analysis and enzyme activity analysis of kiwifruit during postharvest ripening.

Kiwifruit is a climacteric fruit, in which the accumulation of flavor substances mainly occurs at the postharvest ripening stage. However, the dynamic changes in metabolite composition remain poorly understood. Here, targeted multi-platform metabolome analysis based on GC-MS and UPLC-MS/MS and enzyme activity analysis were performed at different postharvest ripening stages of kiwifruit. A total of 12 soluble sugars and 31 organic acids were identified. The main soluble sugars are sucrose, glucose and fructose, which exhibited similar variation tendencies along with the extension of ripening. The main organic acids are citric acid, quinic acid and malic acid, which showed different variation patterns. A total of 48 energy metabolites were identified, which were classified into two groups based on the content variation. The content of substances related to the respiratory metabolic pathway decreased gradually along with postharvest ripening, and there was obvious accumulation of downstream products such as amino acids at the late ripening stage. A total of 35 endogenous hormones were identified, among which seven cytokinins were highly accumulated at the later stage of softening. We further investigated the dynamic changes in the activities of 28 ripening-related enzymes. As a result, the activities of 13 enzymes were highly correlated with changes in starch, total pectin, and soluble sugars, and those of seven enzymes were closely associated with the change in firmness. In conclusion, this study comprehensively describes the dynamic changes in soluble sugars, organic acids, hormones, energy substances, and ripening-related enzyme activities during kiwifruit postharvest ripening, and provides a theoretical basis for the postharvest quality improvement of kiwifruit.

Mycological evaluation of frozen meat with special reference to yeasts.

Fungi can play beneficial and detrimental roles in meat products; however, the diversity and significance of fungi in meat products are poorly understood. This study aimed to isolate and characterize fungal species from frozen beef samples collected from retail stores in the Qena Governorate, Egypt. A total of 70 frozen beef samples were collected from retail stores in Qena, Egypt. All samples were subjected to mycological examination. Fungal colonies were identified using conventional approaches, as well as the VITEK 2 system and DNA sequencing of the internal transcribed spacer region. Analyses of enzymatic activity, biofilm formation ability, and the antimicrobial resistance profiles of the isolated yeasts were also conducted. Molds and yeasts were isolated from 40% and 60% of meat samples, respectively. Mold isolates were dominated by Aspergillus, Penicillium, and Cladosporium spp., whereas yeast isolates were identified as Candida albicans, Candida parapsilosis, Yarrowia lipolytica, Saccharomyces cerevisiae, and Rhodotorula mucilaginosa. Compared to other yeast species, the highest production of lipase and protease was observed in Candida species. The strongest ability to form biofilms was observed in Candida spp., followed by S. cerevisiae, Y. lipolytica, and R. mucilaginosa. The results of antimicrobial susceptibility testing revealed that all yeast isolates showed notable resistance to fluconazole and itraconazole. A significant correlation between antimicrobial resistance and biofilm formation was observed in several species. This study highlights the importance of the dangers of yeasts in food products and the extent of their impact on public health.

Functional identification of anthocyanin glucosyltransferase genes: a Ps3GT catalyzes pelargonidin to pelargonidin 3-O-glucoside painting the vivid red flower color of Paeonia.

Glycosylation from an anthocyanidin 3-O-glucosyltransferase Ps3GT (PsUGT78A27) facilitates the accumulation of pelargonidin 3-O-glucoside, which defines the vivid red flower color and occurs only in specific peony tree cultivars. Although tree peony cultivars of Chinese and Japanese both originated from China, vivid red color is only found in flowers of Japanese cultivars but not of Chinese cultivar groups. In this study, a Japanese tree peony cultivar 'Taiyoh' with vivid red petals and a Chinese tree peony cultivar 'Hu Hong' with reddish pink petals were chosen as the experimental materials. Flavonoids profiling indicated that pelargonidin 3-O-glucoside (Pg3G) detected only in Japanese cultivar contributed to vivid red color of tree peony petals, while pelargonidin 3,5-di-O-glucoside (Pg3G5G) found in both of Japanese and Chinese cultivars was responsible for pink flower color. Through the integration of full-length transcriptome sequencing and in vitro enzymatic activity analysis, two anthocyanin glucosyltransferase genes PsUGT78A27 and PsUGT75L45 were isolated from the petals of tree peony, and their encoding products exhibited enzymatic activities of pelargonidin 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively. Further quantitative real-time PCR revealed that PsUGT78A27 displayed high expression in petals of both cultivars and PsUGT75L45 was expressed at high levels in cultivar 'Hu Hong' only. Using a gene gun technique, the GFP fusion proteins of PsUGT78A27 and PsUGT75L45 were visualized to be cytoplasmic and nuclear localization in the epidermal cells of tree peony petals, and the glucosylation function of PsUGT78A27 and PsUGT75L45 to alter petal color of tree peony and herbaceous peony had been directly validated in vivo. These results demonstrated that PsUGT78A27 and PsUGT75L45 are key players for the presence or absence of vivid red flower color in tree peony cultivars. Our findings further elucidated the chemical and molecular mechanism of petal pigmentation of Paeonia and could help breed the Paeonia cultivars possessing novel flower colors.