Uridyltransferase Research Articles

Metabolic Engineering of Escherichia coli for Hyperoside Biosynthesis.

Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L−1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L−1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L−1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L−1·h−1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.

OP005: AT-007 significantly reduces toxic galactitol in ACTION-galactosemia kids - the 1st therapeutic interventional clinical trial in children with classic galactosemia

Classic Galactosemia (CG) is a rare, autosomal recessive disease where galactose is not metabolized properly due to severe deficiency or absence of the GALT enzyme (galactose-1-phosphate uridyl transferase). At abnormally high levels, galactose becomes an aberrant substrate for the enzyme aldose reductase, resulting in conversion to an abnormal and toxic metabolite, galactitol. Newborn screening and implementation of a galactose-restricted diet has reduced newborn acute symptoms and significantly decreased fatalities. However, despite early dietary intervention, children continue to develop significant morbidities in speech, cognition, behavior, and motor function, which have been shown to progressively worsen with age, as well as cataracts and primary ovarian insufficiency in females. AT-007 is an oral CNS penetrant aldose reductase inhibitor, which prevents conversion of galactose to toxic galactitol, that is in development for the treatment of CG. Reduction in circulating galactitol by AT-007 has been shown to halt the progression of disease in a rat model of Galactosemia. ACTION-Galactosemia Kids is a sequential, two-part, randomized double-blind, placebo-controlled study evaluating the clinical benefit, safety, pharmacokinetics and pharmacodynamics of AT-007 in pediatric patients with CG. Patients were randomized 2:1 to AT-007 or placebo. Disease demographics, genetic variant, and patient history were assessed at baseline. Additionally, functional tests of speech, motor function, cognition and behavior were performed at baseline for all patients enrolled in the clinical study. The primary biomarker study endpoint was reduction in plasma galactitol over a minimum of 30 days of treatment with the target dose of AT-007 vs placebo. Baseline and 30-day plasma galactitol levels were drawn under fasted conditions and evaluated via a validated liquid chromatography-mass spectrometry assay. Baseline galactitol correlation with disease severity was evaluated via a linear model incorporating baseline speech, cognition, behavior and motor skills assessments. Forty-seven patients ranging in age from 2 to 17, were enrolled at 3 US clinical sites in ACTION-Galactosemia Kids across three predefined age groups (2-6 years old; 7-12 years old; 13-17 years old). The patients were predominantly White (98%), had a mean age of 9 and 51% were female/49% male. The most common baseline disease characteristics included learning disorders (∼50%), apraxia (∼40%), anxiety (∼35%), vitamin deficiency (∼30%), tremor (∼20%) and ataxia (∼20%). All the female patients older than 12 had primary ovarian insufficiency. Additionally, ∼10% of the patients suffered from seizures and ∼5% had early onset cataracts. Baseline disease and demographic characteristics were balanced across the placebo and AT-007 treatment groups. Galactitol levels at baseline correlated with total disease severity in this pediatric patient population (p=0.004). Treatment with AT-007 resulted in a 40.2% reduction in galactitol, which was significant when compared with patients on placebo (p<0.001). The reduction in toxic galactitol was consistent among age and dose groups and was not associated with changes in galactose or galacose-1-phosphate (Gal-1p). AT-007 was safe and well tolerated. The biomarker results from ACTION-Galactosemia Kids demonstrates that treatment with AT-007 resulted in a significant reduction of the toxic metabolite galactitol, a biomarker that correlated with disease severity, in pediatric patients with Classic Galactosemia. Treatment of pediatric Galactosemia patients age 2 to 17 with AT-007 was safe and well tolerated.

Neonatal GALT gene replacement offers metabolic and phenotypic correction through early adulthood in a rat model of classic galactosemia

Classic galactosemia (CG) results from profound deficiency of galactose‐1‐P uridylyltransferase (GALT). Despite early detection by newborn screening and lifelong dietary restriction of galactose, most patients grow to experience a range of long‐term complications. Recently, we developed and characterized a GALT‐null rat model of CG and demonstrated that AAV9‐hGALT, administered by tail vein injection to neonatal pups, dramatically improved plasma, liver, and brain galactose metabolites at 2 weeks posttreatment. Here we report a time‐course study of GALT restoration in rats treated as neonates with scAAV9‐hGALT and harvested at 8, 14, 30, and 60 days. Cohorts of rats in the two older groups were weaned to diets containing either 1% or 3% of calories from galactose. As expected, GALT activity in all treated animals peaked early and then diminished over time, most notably in liver, ostensibly due to dilution of the nonreplicating episomal vector as transduced cells divided. All treated rats showed dramatic metabolic rescue through 1 month, and those weaned to the lower galactose diet showed continued strong metabolic rescue into adulthood (2 months). Prepubertal growth delay and cataracts were both partially rescued by treatment. Finally, we found that UDP glucose pyrophosphorylase (UGP), which offers a metabolic bypass around missing GALT, was 3‐fold more active in brain samples from adult rats than from young pups, offering a possible explanation for the improved ability of older GALT‐null rats to metabolize galactose. Combined, these results document promising metabolic and phenotypic efficacy of neonatal GALT gene replacement in a rat model of classic galactosemia.

Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase, UDP-glucose pyrophosphorylase) physiologically aggregated within inclusion bodies retaining their biological activity as immobilized biocatalysts.

Targeting cancer’s sweet spot: UGP2 as a therapeutic vulnerability

ABSTRACT Understanding the mechanisms governing metabolic reprogramming that underlie potential vulnerabilities in cancer cells is key to developing novel therapeutic strategies. The catalytic enzyme UDP-glucose pyrophosphorylase 2 (UGP2) drives the production of UDP-glucose. Our recent work demonstrated the crucial role of UGP2 in cancer growth and its regulation of cellular metabolic processes.

Comparative Transcriptome Analysis of Male Sterile Anthers Induced by High Temperature in Wheat (Triticum aestivum L.).

Global warming will have a negative effect on agricultural production as high temperature (HT) stress can seriously threaten plant growth and reproduction. Male sterility caused by HT may be exploited by the creation of a male-sterile line, which has great potential for application in crop heterosis. Therefore, it is important to understand the molecular mechanisms of anther abortion induced by HT in wheat, which remain unclear at present. In this study, we performed phenotype improve language in the abstract and comparative transcriptome analysis of the male sterile anthers induced by HT in wheat. Compared with Normal anthers, the cytological analysis indicated that HT-induced male sterile anthers were smaller and had no starch accumulation in pollen grains, which is consistent with the results observed by scanning electron microscopy (SEM). The 9601 differentially expressed genes (DEGs) identified by transcriptome sequencing compared with the Normal anthers were noticeably involved in the following pathways: starch and sucrose metabolism, phosphatidylinositol (PI) signaling system, peroxidase activity and response to oxidative stress, and heme binding. In addition, TUNEL assays were performed and the results further confirmed the excessive accumulation of reactive oxygen species (ROS) in sterile anthers. Moreover, a total of 38 hub genes were obtained from the protein-protein interaction network analysis of these pathways, including genes, for example, heat shock protein 90 (HSP90), thioredoxin-like protein 1, peroxidase (POD), calreticulin, UDP glucose pyrophosphorylase (UGPase), sucrose synthase, phosphatidylinositol-4-phosphate 5-Kinase (PIP5K), cytochrome c, and Cystathionine beta-synthase X6-like (CBSX6-like). These findings provide insights for predicting the functions of the candidate genes, and the comprehensive analysis of our results is helpful for studying the abortive interaction mechanism induced by HT in wheat.

Study on the Hepatoprotection of Schisandra chinensis Caulis Polysaccharides in Nonalcoholic Fatty Liver Disease in Rats Based on Metabolomics.

The aim of this study was to investigate the hepatoprotection of Schisandra chinensis Caulis polysaccharides (SCPs) in the nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) in rats. A total of 30 Wistar rats were randomly divided into the control group (CON), model group (MOD), and Schisandra chinensis caulis polysaccharide (SCP) group. Except for those in the CON group, the other rats were fed with high-fat diet for 4 weeks to establish an NAFLD model. From the 5th week, rats in the SCP group were given SCP solution (100 mg kg−1) by gavage for 6 weeks, and those in the CON and MOD groups were given an equal volume of distilled water in the same way. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) levels in serum, the malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities in the liver tissue were detected. The small molecular metabolites in the blood of rats were determined by the metabolomics method of ultra-high-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) combined with multivariate analysis. The enrichment analysis and pathway analysis of the different metabolites were carried out. The therapeutic mechanism of SCP in NAFLD rats was verified by western blot. The results showed that the levels of AST, ALT, TG, TC, and LDL-C in the serum of rats in the SCP group were significantly lower, and the levels of HDL-C were significantly higher than those in the MOD group. The screening and analysis of the metabolic pathways showed that SCP could alleviate the development of NAFLD by regulating the expression of UDP-glucose pyrophosphorylase (UGP2), UDP-glucose 6-dehydrogenase (UGDH), acetyl CoA carboxylase (ACC), and fatty acid synthase (FAS) in the liver of NAFLD rats. This study may provide a theoretical basis for the development and utilization of SCP.

Engineering of continuous bienzymatic cascade process using monolithic microreactors – In flow synthesis of trehalose

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 gproduct·L-1·h−1·mgprotein-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

Transcriptome analysis reveals the metabolisms of starch degradation and ethanol fermentation involved in alcoholic off-flavour development in kiwifruit during ambient storage

Kiwifruit tends to develop alcoholic off-flavour after long-term storage, leading to decreased fruit market value. Actinidia eriantha ‘White’ and Actinidia deliciosa ‘Bruno’ are two major cultivated kiwifruits in China. Compared to A. eriantha ‘White’, A. deliciosa ‘Bruno’ is prone to developing alcoholic off-flavour associated with ethanol accumulation during storage at ambient conditions without any stresses. In this study, to better understand the complex mechanisms responsible for developing alcoholic off-flavour in kiwifruit during postharvest, transcriptome analysis was used to compare the difference in metabolic pathways between kiwifruits ‘white’ and ‘Bruno’ during ripening at ambient storage. Our results indicated that the development of alcoholic off-flavour in kiwifruit ‘Bruno’ might be associated with three metabolic pathways: the respiration and ethylene metabolisms, starch and sucrose metabolism and ethanol fermentation metabolism. In kiwifruit ‘Bruno’, the higher respiration rate and ethylene production might increase the energy conversion and internal carbon dioxide (CO2) accumulation. Moreover, the higher expressions of starch phosphorylases (SPs), beta-amylases, UDP-glucose pyrophosphorylases (UGPases), sucrose synthases (SSs) and invertases (INVs) accelerated starch degradation along with soluble sugars accumulation in kiwifruit ‘Bruno’, which could provide adequate substrates for ethanol fermentation. In addition, the higher activity of ethanol fermentation metabolism associated with higher expressions of pyruvate kinases (PKs), NADP-dependent malic enzymes (NADP-MEs), pyruvate decarboxylases (PDCs) and alcohol dehydrogenases (ADHs) might directly increase the ethanol production.

Global Analysis of UDP Glucose Pyrophosphorylase (UDPGP) Gene Family in Plants: Conserved Evolution Involved in Cell Death.

UDP glucose pyrophosphorylase (UDPGP) family genes have been reported to play essential roles in cell death or individual survival. However, a systematic analysis on UDPGP gene family has not been performed yet. In this study, a total of 454 UDPGP proteins from 76 different species were analyzed. The analyses of the phylogenetic tree and orthogroups divided UDPGPs into three clades, including UDP-N-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase (UGP, containing UGP-A and UGP-B), and UDP-sugar pyrophosphorylase (USP). The evolutionary history of the UDPGPs indicated that the members of UAP, USP, and UGP-B were relatively conserved while varied in UGP-A. Homologous sequences of UGP-B and USP were found only in plants. The expression profile of UDPGP genes in Oryza sativa was mainly motivated under jasmonic acid (JA), abscisic acid (ABA), cadmium, and cold treatments, indicating that UDPGPs may play an important role in plant development and environment endurance. The key amino acids regulating the activity of UDPGPs were analyzed, and almost all of them were located in the NB-loop, SB-loop, or conserved motifs. Analysis of the natural variants of UDPGPs in rice revealed that only a few missense mutants existed in coding sequences (CDSs), and most of the resulting variations were located in the non-motif sites, indicating the conserved structure and function of UDPGPs in the evolution. Furthermore, alternative splicing may play a key role in regulating the activity of UDPGPs. The spatial structure prediction, enzymatic analysis, and transgenic verification of UAP isoforms illustrated that the loss of N- and C-terminal sequences did not affect the overall 3D structures, but the N- and C-terminal sequences are important for UAP genes to maintain their enzymatic activity. These results revealed a conserved UDPGP gene family and provided valuable information for further deep functional investigation of the UDPGP gene family in plants.

Hormone-Sensitive Gene Signatures in the Mammary Epithelial Cells of Lactating Women With Persistent Low Milk Production

ObjectivesInsulin resistance may contribute to the association between maternal obesity and lactation difficulties. Our objective was to examine differential expression of genes involved in insulin-sensitive and prolactin-sensitive signaling in the mammary glands of women with very low, versus sufficient, milk production. MethodsAmong a subset of mothers in a study of low milk supply, extracellular mammary epithelial cell mRNA was isolated from fresh milk fat and submitted for RNA-sequencing. Aligned and quantified reads were examined for differentially expressed genes by t-test (DEG, p < 0.05 after false discovery rate adjustment). Insulin signaling and prolactin signaling KEGG pathways, in addition to a hand-curated gene list developed from relevant literature, were used for targeted DEG analysis. Sample size (milk volume range) for the sampled breast was n = 5 for the lowest milk production group [LMP] (0–53 mL/24 h), versus n = 4 for the highest milk production group [HMP] (422–463 mL/24 h). Clinical measures of metabolic health included BMI and fasting insulin and glucose. ResultsAll LMP samples were from mothers with obesity and with significantly elevated markers of insulin resistance versus the HMP group (e.g., HOMA-IR 1.7 versus 1.1, p < 0.05). DEG in the LMP group revealed significantly lower insulin-sensitive signaling, including a 2.3-fold decrease in insulin receptor substrate 2 (IRS2), versus HMP. There was no differential expression for genes involved in prolactin signaling, such as the prolactin receptor or JAK2. In LMP versus HMP groups, synthesis of beta-casein (CSN2) was 2.1-fold lower; expression of acetyl-CoA carboxylase 2 (ACACB), a gene downstream of insulin signaling that inhibits fatty acid oxidation, was 3.1-fold lower; and expression of UDP-glucose pyrophosphorylase 2 (UGP2), a rate-limiting gene in lactose synthesis and thus a determinant of milk volume, was 2.7-fold lower. ConclusionsSuppressed milk production by obese, LMP mothers is associated with downregulation of insulin-signaling genes but not hallmark prolactin-signaling genes. Overall, gene expression in the LMP group portrays an insulin-resistant signature in the mammary gland, with downregulation of insulin-sensitive anabolic processes and upregulation of catabolic gene expression. Funding SourcesNIH.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

ABSTRACTGlycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography–high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones.

Salt-Tolerant Synechococcus elongatus UTEX 2973 Obtained via Engineering of Heterologous Synthesis of Compatible Solute Glucosylglycerol.

The recently isolated cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) is characterized by a faster growth rate and greater tolerance to high temperature and high light, making it a good candidate chassis for autotrophic photosynthetic microbial cell factories. However, Syn2973 is sensitive to salt stress, making it urgently important to improve the salt tolerance of Syn2973 for future biotechnological applications. Glucosylglycerol, a compatible solute, plays an important role in resisting salt stress in moderate and marine halotolerant cyanobacteria. In this study, the salt tolerance of Syn2973 was successfully improved by introducing the glucosylglycerol (GG) biosynthetic pathway (OD750 improved by 24% at 60 h). In addition, the salt tolerance of Syn2973 was further enhanced by overexpressing the rate-limiting step of glycerol-3-phosphate dehydrogenase and downregulating the gene rfbA, which encodes UDP glucose pyrophosphorylase. Taken together, these results indicate that the growth of the end-point strain M-2522-GgpPS-drfbA was improved by 62% compared with the control strain M-pSI-pSII at 60 h under treatment with 0.5 M NaCl. Finally, a comparative metabolomic analysis between strains M-pSI-pSII and M-2522-GgpPS-drfbA was performed to characterize the carbon flux in the engineered M-2522-GgpPS-drfbA strain, and the results showed that more carbon flux was redirected from ADP-GLC to GG synthesis. This study provides important engineering strategies to improve salt tolerance and GG production in Syn2973 in the future.

Disruption of carbohydrate and proline metabolism in anthers under low temperature causes pollen sterility in chickpea

Chickpea (Cicer arietinum L.) is the third most important food legume globally and is an important source of high-quality proteins to both humans and animals. It is highly sensitive to low temperature (LT) resulting in large scale yield losses. LT induces flower abortion in chickpea by disruption of gamete development and induction of pollen sterility. However, molecular mechanisms governing LT induced pollen sterility in chickpea are unknown. Disruption of carbohydrate metabolism has been implicated in LT induced damage to crops whereas proline and antioxidants protect plants from LT stress. In this study, the impact of LT on carbohydrates, proline and enzymatic antioxidants and expression of genes of these metabolite pathways were investigated in anthers at vacuolated pollen stage. The study included two genotypes, a cold-sensitive (GPF2) and a cold-tolerant (ICC 16349) that differed for pollen sterility under LT. Metabolite profiling revealed a reduction in starch and proline content in GPF2 under LT, whereas these parameters did not change but non-reducing sugars increased in ICC 16349. At the transcriptional level, cold initially lowered the transcription of all genes (19) under study in GPF2 and 14 genes out of 19 in ICC 16349. Initial down-regulation was followed by differential reactivation of transcription machinery in GPF2 and ICC 16349 at 30 min. Under LT, expression of starch synthesis, starch degradation, sucrose synthesis and sucrose transport genes showed a contrasting response in the two genotypes, where GPF2 showed downregulation of these genes while ICC 16,349 showed upregulation. The proline transport gene (proline transporter 1) showed higher downregulation in GPF2 than ICC 16349. Overall, pollen viability under LT in ICC 16349 was linked to the maintenance of starch, reducing sugars and proline levels and over-expression of UDP glucose pyrophosphorylase and cell wall invertase, while these processes were compromised in the GPF2 possibly leading to pollen sterility.

UDP-Glucose Pyrophosphorylase ugp1 is Required in Heat Stress Response of Saccharomyces cerevisiae

UDP-glucose pyrophosphorylase (UGP1) is an important metabolic enzyme in Saccharomyces cerevisiae. In this research, we built a strain with reduced ugp1 level for exploring its biological roles. The survival rate, trehalose, glucan, and the sensitivity to cell wall perturbing agents were tested. Intracellular trehalose and glucan of ugp1 knockdown (ugp1KD) strain showed 37.4% and 56.69% of WT strain at 42 °C. Meanwhile, the survival rate declined to 33.83% after 2 hours and to 8.44% after 8 hours. The cell wall was more susceptible to Congo red, SDS, and Calcofluor white. After complementing ugp1(UGP1C), the resistance to heat-stress and cell wall perturbing agents were rescued markedly. The 1,3-β-D-glucan synthase (FKS1) expression level was affected by knocking down ugp1, as well. Our data suggest that ugp1 is required in the heat stress response of S. cerevisiae by influencing trehalose and glucan, and provides a foundation elucidating the essence of survival under heat stress.

Elucidating carbohydrate metabolism in Euglena gracilis: Reverse genetics-based evaluation of genes coding for enzymes linked to paramylon accumulation

Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear β-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176–186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 μg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway’s functionality in Euglena gracilis.

An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation.

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we have isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity, likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

Transcriptome Analysis Reveals Photoperiod-Associated Genes Expressed in Rice Anthers.

Environmental conditions, such as photoperiod and temperature, can affect male fertility in plants. While this feature is heavily exploited in rice to generate male-sterile lines for hybrid breeding, the underlying molecular mechanisms remain largely unknown. In this study, we use a transcriptomics approach to identify key genes and regulatory networks affecting pollen maturation in rice anthers in response to different day lengths. A total of 11,726 differentially expressed genes (DEGs) were revealed, of which 177 were differentially expressed at six time points over a 24-h period. GO enrichment analysis revealed that genes at all time points were enriched in transport, carbohydrate, and lipid metabolic processes, and signaling pathways, particularly phytohormone signaling. In addition, co-expression network analysis revealed four modules strongly correlated with photoperiod. Within these four modules, 496 hub genes were identified with a high degree of connectivity to other photoperiod-sensitive DEGs, including two previously reported photoperiod- and temperature-sensitive genes affecting male fertility, Carbon Starved Anther and UDP-glucose pyrophosphorylase, respectively. This work provides a new understanding on photoperiod-sensitive pollen development in rice, and our gene expression data will provide a new, comprehensive resource to identify new environmentally sensitive genes regulating male fertility for use in crop improvement.

Overexpression of DoUGP Enhanced Biomass and Stress Tolerance by Promoting Polysaccharide Accumulation in Dendrobium officinale.

Uridine diphosphate glucose pyrophosphorylase (UDP-glucose pyrophosphorylase, UGPase), as one of the key enzymes in polysaccharide synthesis, plays important roles in the growth and development of plants. In this study, the DoUGP gene of Dendrobium officinale was overexpressed. The expression of DoUGP and genes playing roles in the same and other saccharide synthesis pathways was determined, and the total soluble polysaccharide was also tested in wild-type and transgenic seedlings. We also performed freezing and osmotic stress treatments to determine whether overexpression of DoUGP could influence stress resistance in transgenic seedlings. Results showed that mRNA expression levels of DoUGP and its metabolic upstream and downstream genes in the transgenic seedlings were increased compared to the expression of these genes in wild-type seedlings. Additionally, most CSLA genes involved in the biosynthesis of mannan polysaccharides were significantly upregulated. The total polysaccharide and mannose content of transgenic seedlings were increased compared to the content of wild type, and enhanced stress tolerance was found in the overexpressed seedlings compared to the wild type.