Uridine Diphosphate Research Articles

Establishment of UGT1A1-knockout human iPS-derived hepatic organoids for UGT1A1-specific kinetics and toxicity evaluation

Uridine diphosphate glucuronosyltransferases (UGTs) are highly expressed in the liver and are involved in the metabolism of many drugs. In particular, UGT1A1 has a genetic polymorphism that causes decreased activity, leading to drug-induced hepatotoxicity. Therefore, an invitro evaluation system that accurately predicts the kinetics of drugs involving UGT1A1 is required. However, there is no such evaluation system because of the absence of the UGT1A1-selective inhibitor. Here, using human induced pluripotent stem (iPS) cells, genome editing technology, and organoid technology, we generated UGT1A1-knockout human iPS hepatocyte-derived liver organoids (UGT1A1-KO i-HOs) as a model for UGT1A1-specific kinetics and toxicity evaluation. i-HOs showed higher gene expression of many drug-metabolizing enzymes including UGT1A1 than human iPS cell-derived hepatocyte-like cells (iPS-HLCs), suggesting that hepatic organoid technology improves liver functions. Wild-type (WT) i-HOs showed similar levels of UGT1A1 activity to primary human (cryopreserved) hepatocytes, while UGT1A1-KO i-HOs completely lost the activity. Additionally, to evaluate whether this model can be used to predict drug-induced hepatotoxicity, UGT1A1-KO i-HOs were exposed to SN-38, the active metabolite of irinotecan, an anticancer drug, and acetaminophen and confirmed that these cells could predict UGT1A1-mediated toxicity. Thus, we succeeded in generating model cells that enable evaluation of UGT1A1-specific kinetics and toxicity.

Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Protein Modelling Highlighted Key Catalytic Sites Involved in Position-Specific Glycosylation of Isoflavonoids.

Uridine diphosphate glycosyltransferases (UGTs) are known for promiscuity towards sugar acceptors, a valuable characteristic for host plants but not desirable for heterologous biosynthesis. UGTs characterized for the O-glycosylation of isoflavonoids have shown a variable efficiency, substrate preference, and OH site specificity. Thus, 22 UGTs with reported isoflavonoid O-glycosylation activity were analyzed and ranked for OH site specificity and catalysis efficiency. Multiple-sequence alignment (MSA) showed a 33.2% pairwise identity and 4.5% identical sites among selected UGTs. MSA and phylogenetic analysis highlighted a comparatively higher amino acid substitution rate in the N-terminal domain that likely led to a higher specificity for isoflavonoids. Based on the docking score, OH site specificity, and physical and chemical features of active sites, selected UGTs were divided into three groups. A significantly high pairwise identity (67.4%) and identical sites (31.7%) were seen for group 1 UGTs. The structural and chemical composition of active sites highlighted key amino acids that likely define substrate preference, OH site specificity, and glycosylation efficiency towards selected (iso)flavonoids. In conclusion, physical and chemical parameters of active sites likely control the position-specific glycosylation of isoflavonoids. The present study will help the heterologous biosynthesis of glycosylated isoflavonoids and protein engineering efforts to improve the substrate and site specificity of UGTs.

Analysis of metabolite differences between South Korean and Chinese yellow goosefish (Lophius litulon) using capillary electrophoresis time-of-flight mass spectrometry

The yellow goosefish is a benthic fish that belongs to the family Lophiidae and order Lophiiformes and is distributed in the Yellow and East China Seas. This study aimed to distinguish between yellow goosefish from different geographical origins by analyzing their metabolites. Capillary electrophoresis time-of-flight mass spectrometry was used to analyze metabolite profiles in the muscle tissues of yellow goosefish to distinguish between Korean and Chinese yellow goosefish. In total, 271 putative metabolites were extracted using 50% acetonitrile in water. Principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to distinguish different geographical origins using the metabolite profiles obtained. The R2 and Q2 values of the OPLS-DA model were 0.856 and 0.695, respectively, indicating that the model was well-fitted and had good predictability. The heat map revealed that nucleic acid and amino compounds differed between the Korean and Chinese fish, and the variable importance in the projection scores obtained from OPLS-DA showed that there were geographical differences in the primary metabolites (5′-methylthioadenosine, adenosine, uridine 5-diphosphate, guanosine 5-diphosphate, urea, homocarnosine, O-acetylcarnitine, cycloleucine, cycloleucine S-adenosylmethionine, S-adenosylhomocysteine, ethanolamine, myo-inositol 1-phosphate), which were identified as potential candidate biomarkers.

Role of the P2Y6 receptor of UDP in the modulation of murine dendritic cell functions and Th1 polarisation of the immune response

Numerous studies have demonstrated the role of uridine diphosphate (UDP) and its P2Y6 receptor in the inflammatory reaction and innate immunity. However, the importance of the P2Y6 receptor in the adaptive immune response remains unclear. In this study, we demonstrate that the P2Y6 receptor is functionally expressed in murine bone marrow dendritic cells (BMDC). UDP induced a Ca2+ transient in these cells that was decreased in P2Y6-deficient mice. UDP also increased the endocytosis of fluorescein isothiocyanate-dextran (FITC-dextran) and amplified the secretion of interleukin 12-p70 (IL-12p70) induced by CpG; these responses were abolished in P2Y6-deficient mice. In vivo experiments showed that the serum level of specific IgG2c after immunisation with ovalbumin was decreased in P2Y6-deficient mice, while the level of specific IgG1 was unchanged. These data suggest that the P2Y6-mediated effects of UDP on myeloid dendritic cells play a role in the in vivo Th1 skewing of the immune response.

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

Construction of a UDP-Arabinose Regeneration System for Efficient Arabinosylation of Pentacyclic Triterpenoids.

Glycosylation is an important method of modifying natural products and is usually catalyzed by uridine 5'-diphosphate (UDP)-glycosyltransferase. UDP-β-l-arabinose (UDP-Ara) confers specific functions to natural products such as pentacyclic triterpenoids. However, UDP-arabinosyltransferase with high regioselectivity toward pentacyclic triterpenoids has rarely been reported. In addition, UDP-Ara is mainly biosynthesized from UDP-α-d-glucose (UDP-Glc) through several reaction steps, resulting in the high cost of UDP-Ara. Herein, UGT99D1 was systematically characterized for specifically transferring one moiety of arabinose to the C-3 position of typical pentacyclic triterpenoids. Subsequently, 15 enzymes from plants, mammals, and microorganisms were characterized, and a four-enzyme cascade comprising sucrose synthase, UDP-Glc dehydrogenase, UDP-α-d-glucuronic acid decarboxylase, and UDP-Glc 4-epimerase was constructed to transform sucrose into UDP-Ara with UDP recycling. This system was demonstrated to efficiently produce the arabinosylated derivative (Ara-BA) of typical pentacyclic triterpenoid betulinic acid (BA). Finally, the in vitro cytotoxicity tests indicated that Ara-BA showed much higher anticancer activities than BA. The established arabinosylation platform shows the potential to enhance the pharmacological activity of natural products.

Combined effect of PNPLA3 rs738409 and UGT1A1 rs10929303 gene polymorphisms on nonalcoholic fatty liver disease in children

Objective: To investigate the combined effects of patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 (C > G) and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) rs10929303 (C > T) on nonalcoholic fatty liver disease (NAFLD) in children and adolescents so as to provide scientific evidence for NAFLD genetic research. Methods: 1 027 children and adolescents aged 7-18 were selected as the research subjects. The general situation, past medical history, height and body weight measurements, and B- mode ultrasound test of the liver were investigated by dedicated full-time personnel. In addition, the morning fasting venous blood was collected to measure the blood biochemical indicators. DNA was extracted and genotyped for PNPLA3 rs738409 and UGT1A1 rs10929303. Logistic regression analysis was used to analyze the association and combined effect of the two gene polymorphisms and NAFLD. Statistical analysis was performed by t-test, Mann-Whitney U test, or c2 test according to different data. Results: The GG genotype of PNPLA3 rs738409 and the CC genotype of UGT1A1 rs10929303 were associated with an increased risk of developing NAFLD in children by 89% (OR = 1.89, 95% CI: 1.11-3.23, P = 0.019) and 96% (OR = 1.96, 95% CI: 1.21-3.17, P = 0.006), respectively, while the concurrent risk of NAFLD in those who carried the above two genotypes increased by 306% compared with those who did not carry both genotypes (OR = 4.06, 95% CI: 1.90 ~ 8.66, P < 0.001). Conclusion: The combined effect of PNPLA3 and UGT1A1 gene polymorphisms can significantly increase the risk of NAFLD in children, providing new evidence for elucidating the genetic susceptibility to NAFLD.

CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function.

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Leveraging glycoside-targeted metabolomics to gain insight into biological function.

In plants, uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) catalyze glycosylation of secondary metabolites, but assigning physiological functions to UGTs is still a daunting task. The recent study of Wu et al. presents a useful method to resolve this problem by elegantly combining modification-specific metabolomics with isotope tracing.

MK8617 inhibits M1 macrophage polarization and inflammation via the HIF-1α/GYS1/UDPG/P2Y14 pathway.

Nonresolving inflammation is a major driver of disease and needs to be taken seriously. Hypoxia-inducible factor (HIF) is closely associated with inflammation. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs), as stabilizers of HIF, have recently been reported to have the ability to block inflammation. We used MK8617, a novel HIF-PHI, to study its effect on macrophage inflammation and to explore its possible mechanisms. Cell viability after MK8617 and lipopolysaccharide (LPS) addition was assessed by Cell Counting Kit-8 (CCK8) to find the appropriate drug concentration. MK8617 pretreated or unpretreated cells were then stimulated with LPS to induce macrophage polarization and inflammation. Inflammatory indicators in cells were assessed by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot (WB) and immunofluorescence (IF). The level of uridine diphosphate glucose (UDPG) in the cell supernatant was measured by ELISA. Purinergic G protein-coupled receptor P2Y14, as well as hypoxia-inducible factor-1α (HIF-1α) and glycogen synthase 1 (GYS1) were detected by qRT-PCR and WB. After UDPG inhibition with glycogen phosphorylase inhibitor (GPI) or knockdown of HIF-1α and GYS1 with lentivirus, P2Y14 and inflammatory indexes of macrophages were detected by qRT-PCR and WB. MK8617 reduced LPS-induced release of pro-inflammatory factors as well as UDPG secretion and P2Y14 expression. UDPG upregulated P2Y14 and inflammatory indicators, while inhibition of UDPG suppressed LPS-induced inflammation. In addition, HIF-1α directly regulated GYS1, which encoded glycogen synthase, an enzyme that mediated the synthesis of glycogen by UDPG, thereby affecting UDPG secretion. Knockdown of HIF-1α and GYS1 disrupted the anti-inflammatory effect of MK8617. Our study demonstrated the role of MK8617 in macrophage inflammation and revealed that its mechanism of action may be related to the HIF-1α/GYS1/UDPG/P2Y14 pathway, providing new therapeutic ideas for the study of inflammation.

Metabolic engineering of Yarrowia lipolytica for high-level production of scutellarin

Scutellarin drugs have been recognized as a key item in the national development of essential clinical emergency drugs for treating cardiovascular and cerebrovascular diseases; therefore, the market demand for scutellarin is growing rapidly. Microbial synthesis based on synthetic biology is a promising method for industrial production of scutellarin. In this study, the highest reported scutellarin titer in the shake flask of 703.01 ± 4.83 mg/L was achieved in Yarrowia lipolytica through the systematic metabolic engineering modifications, including screening for the optimal flavone-6-hydroxylase–cytochrome P450 reductase combination SbF6H–ATR2 to enhance P450 enzyme activity, increasing the copy numbers of rate-limiting enzyme genes, overexpressing ZWF1 and GND1 to increase NADPH supply, enhancing the supply of p-coumaric acid and uridine diphosphate glucose, and introducing the heterologous gene VHb to enhance oxygen supply. This study has significant implications for the industrial production of scutellarin and other valuable flavonoids in green economies.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Fetal Reprogramming of Nutrient Surplus Signaling, O-GlcNAcylation, and the Evolution of CKD.

Fetal kidney development is characterized by increased uptake of glucose, ATP production by glycolysis, and upregulation of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 alpha (HIF-1 α ), which (acting in concert) promote nephrogenesis in a hypoxic low-tubular-workload environment. By contrast, the healthy adult kidney is characterized by upregulation of sirtuin-1 and adenosine monophosphate-activated protein kinase, which enhances ATP production through fatty acid oxidation to fulfill the needs of a normoxic high-tubular-workload environment. During stress or injury, the kidney reverts to a fetal signaling program, which is adaptive in the short term, but is deleterious if sustained for prolonged periods when both oxygen tension and tubular workload are heightened. Prolonged increases in glucose uptake in glomerular and proximal tubular cells lead to enhanced flux through the hexosamine biosynthesis pathway; its end product-uridine diphosphate N -acetylglucosamine-drives the rapid and reversible O-GlcNAcylation of thousands of intracellular proteins, typically those that are not membrane-bound or secreted. Both O-GlcNAcylation and phosphorylation act at serine/threonine residues, but whereas phosphorylation is regulated by hundreds of specific kinases and phosphatases, O-GlcNAcylation is regulated only by O-GlcNAc transferase and O-GlcNAcase, which adds or removes N-acetylglucosamine, respectively, from target proteins. Diabetic and nondiabetic CKD is characterized by fetal reprogramming (with upregulation of mTOR and HIF-1 α ) and increased O-GlcNAcylation, both experimentally and clinically. Augmentation of O-GlcNAcylation in the adult kidney enhances oxidative stress, cell cycle entry, apoptosis, and activation of proinflammatory and profibrotic pathways, and it inhibits megalin-mediated albumin endocytosis in glomerular mesangial and proximal tubular cells-effects that can be aggravated and attenuated by augmentation and muting of O-GlcNAcylation, respectively. In addition, drugs with known nephroprotective effects-angiotensin receptor blockers, mineralocorticoid receptor antagonists, and sodium-glucose cotransporter 2 inhibitors-are accompanied by diminished O-GlcNAcylation in the kidney, although the role of such suppression in mediating their benefits has not been explored. The available evidence supports further work on the role of uridine diphosphate N -acetylglucosamine as a critical nutrient surplus sensor (acting in concert with upregulated mTOR and HIF-1 α signaling) in the development of diabetic and nondiabetic CKD.

Ozone mitigates the flesh discoloration in response to 1-methylcyclopropene by promoting anthocyanin biosynthesis in postharvest nectarines

The effects of combined treatments with 1-methylcyclopropene (1-MCP) and ethylene absorbent (EA) or ozone (O3) on the anthocyanins biosynthesis in nectarines stored at 0 °C were investigated during the storage period. The results indicated that 1-MCP+EA and 1-MCP+O3 effectively inhibited respiration and ethylene release. 1-MCP+EA reduced anthocyanins accumulation and inhibited the red coloration in pulp, whereas 1-MCP+O3 accelerated the red pigmentation. Contrasted to the 1-MCP group, 1-MCP+O3 exhibited higher anthocyanins contents, expression levels of PpPAL1, PpCHS, PpDFR, PpANS, PpUFGT, and PpMYB10.2 and activities of 4-coumarate coenzyme A ligase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, uridine diphosphate flavonoid 3-O-glucosyltransferase involved in the phenylpropanoid pathway. These results indicated that 1-MCP+O3 promoted the anthocyanins biosynthesis due to the up-regulation of enzymes activities and anthocyanins biosynthetic genes and transcription factor. This study may shed new light on improving quality and color development and optimizing 1-MCP application for postharvest preservation of fruit.

Identification of UGT85A glycosyltransferases associated with volatile conjugation in grapevine (Vitis vinifera × Vitislabrusca)

Glycosylated volatiles are considered as hidden aroma precursors that affect fruit flavor and are involved in plant defense response. Several uridine-diphosphate (UDP)-glycosyltransferase (UGT) members related to the glycosylation of volatiles have been identified from grapevine berries. Although grapevine leaves produced much higher levels of glycosylated volatiles than berries, UGTs responsible for the production of glycosylated volatiles in leaves have not yet been identified. Here, eight novel UGT genes were cloned, among these four members that belong to UGT85A family (named UGT85A24, UGT85A25, UGT85A26, and UGT85A27) were localized in the cytoplasm and their recombinant proteins showed activity toward volatiles detected in grapevine, with geraniol as the preferred substrate in vitro. Two UGT genes with 98.1% homology differ in R/S-linalool selection. The UGT85A26-catalyzed substrate showed enrichment in S-linalool, whereas UGT85A27 showed a strong preference for R-linalool. UGT85A24 and UGT85A25 were expressed mainly in berries, and UGT85A26 and UGT85A27 were expressed predominantly in grapevine mature leaves and correlated with the highest content of glycosylated volatiles in the mature leaves. Methyl jasmonate (MeJA) induced the expression of UGT85A26 and UGT85A27 as well as the production of linalyl-β-glucoside, citronellyl-β-glucoside and phenethyl-β-glucoside. Transient overexpression of UGT85A26 and UGT85A27 in tobacco leaves led to the accumulation of linalyl-β-glucoside and an increase in citronellyl-β-glucoside in vivo. These results indicate that the expression of UGT85A26 and UGT85A27 is a major regulator affecting the content of glycosylated volatiles in grapevine.

Conserved active site architecture between bacterial cellulose and chitin synthases.

Glycosyltransferases (GTs) are a large and diverse group of enzymes responsible for catalyzing the formation of a glycosidic bond between a donor molecule, usually a monosaccharide, and a wide range of acceptor molecules, thus, playing critical roles in various essential biological processes. Chitin and cellulose synthases are two inverting processive integral membrane GTs, belonging to the type-2 family involved in the biosynthesis of chitin and cellulose, respectively. Herein, we report that bacterial cellulose and chitin synthases share an E-D-D-ED-QRW-TK common motif spatially co-localized among distant bacterial evolutionary species despite their low amino acid sequence and structural similarities. This theoretical framework offers a new perspective to the current view that bacterial cellulose and chitin synthases are substrate specific and that chitin and cellulose are organism specific. It lays the ground for future in vivo and in silico experimental assessment of cellulose synthase catalytic promiscuity against uridine diphosphate N-acetylglucosamine and chitin synthase against uridine diphosphate glucose, respectively.

Characterization of UDP‐glycosyltranferase genes and their roles in chlorfenapyr tolerance in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

AbstractGlyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the main pest of mulberry leaves and causes great losses to the sericultural industry in China. Chlorfenapyr is a new type of arylpyrrole insecticide in mulberry fields. Uridine diphosphate–glycosyltransferase (UGT) is the major phase II metabolizing enzymes that play important roles in detoxifying the toxic substances in insects. However, the roles of UGT genes against chemical insecticides in lepidopteran pests still receive less attention. To better understand the mechanism of insecticide tolerance in G. pyloalis, a LC50 of chlorfenapyr to the fourth‐instar larvae was firstly determined. Subsequently, 24 UGT genes were identified from the previous constructed larvae transcriptome of G. pyloalis. Among these, 23 contained full‐length ORFs and shared high homology with Ostrinia furnacalis and Chilo suppressalis. qRT‐PCR validation revealed that four GpylUGTs (GpUGT33AQ1, GpUGT33AS1, GpUGT50A14, and GpUGT40AV1) were significantly upregulated after 24 h of chlorfenapyr treatment. Three GpylUGTs (GpUGT33AS1, GpUGT40AM4, and GpUGT40AK7) were upregulated after 48 h of chlorfenapyr treatment compared to the control groups. To further explore the functions of UGT genes, GpUGT33AS1 was selected and knocked down, which resulted in the decline of UGT expression and significantly increased the mortality of G. pyloalis larvae under LC30 chlorfenapyr exposure. This study revealed that UGTs in G. pyloalis contributed to the tolerance towards chlorfenapyr and provided basic insight into the insecticide detoxification mechanism in lepidopteran pests.