UDP-glucose Pyrophosphorylase Gene Research Articles

Why study the UGP gene in grafted woody plants?

Grafting is a technique for cultivating commercial fruit species that guarantees genetic characteristics using selected clones. However, candidate genes involved in the re-establishment of grafted woody plants remain poorly understood. In this way, our manuscript aimed to present current knowledge on UDP-glucose pyrophosphorylase (UGP) gene expression and its enzymatic activity (UGPase) by incorporating Brazilian wild to elucidate the role of “players” in re-establishing post-grafting in woody species. Considerable benefits were observed in grafted plants, explained by the correlation between gene expression, such as that of UGP, and UGPase biosynthesis and enzymatic activity. This enzyme is directly responsible for cell wall formation, which explains faster tissue formation in some grafting combinations than others. Besides, UGP promotes the re-establishment of the vascular connection in plant grafting, thus regulating short- and long-term grafting compatibility in woody plants. These crucial experiments are also highly relevant to many nurseries. In this study, we report that the Annona genus is a valuable plant model for studying plant grafting in wood species.

Increased production and anti-senescence activity of exopolysaccharides in Ganoderma lingzhi by co-overexpression of β-1,3-glucan synthase and UDP-glucose pyrophosphorylase

A β-1,3-glucan synthase gene (gls) was cloned and overexpressed in Ganoderma lingzhi. The content of intracellular polysaccharides (IPS) in G. lingzhi overexpressing gls was 22.36 mg/100 mg dry weight (DW), 19 % higher than those in the wild-type (WT) strain. Overexpression of gls did not affect the expression of the phosphoglucomutase gene and the UDP-glucose pyrophosphorylase gene (ugp) in the polysaccharide biosynthesis. The gls and ugp were then simultaneously overexpressed in G. lingzhi for the first time. The combined overexpression of these two genes increased the IPS content and exopolysaccharides (EPS) production to a greater extent than the overexpression of gls independently. The maximum IPS content of the overexpressed strain was 24.61 mg/100 mg, and the maximum EPS production was 1.55 g/L, 1.31- and 1.50-fold higher than that in the WT strain, respectively. Moreover, the major EPS fractions from the overexpression strain contained more glucose (86.7 % and 72.5 %) than those from the WT strain (78.2 % and 62.9 %). Furthermore, the major fraction G+U-0.1 from the overexpression strain exhibited stronger antioxidant and anti-senescence activities than the WT-0.1 fraction from the WT strain. These findings will aid in the hyperproduction and application of Ganoderma polysaccharides and facilitate our understanding of mushroom polysaccharide biosynthesis.

An emerging concept of diurnal variation in expression of UDP-Glucose Pyrophosphorylase gene, (StUGPase2), in potato (Solanum tuberosum L.)

Diurnal regulation of gene expression encoding the dominant isoform of UDP-Glucose Pyrophosphorylase, StUGPase2, was studied in potato leaves at different developmental stages (pre-tuberization, tuber initiation, and tuber bulking). Across the developmental stages, the StUGPase2 steady-state mRNA level increased gradually from morning (6 AM) to noon (12 PM), followed by a transient drop at 2 PM. The mRNA level increased thereafter, and the highest transcript abundance was recorded at 8:00 PM. A sharp decline in StUGPase2 transcript abundance at 2 PM in all the stages studied is believed to be associated with the previous reports of diurnal behaviour of sucrose concentration and light responsiveness of sucrose synthase enzyme. Moreover, in all three stages, a steady state increase of StUGPase2 transcript despite a consistent decrease in irradiance after 12 PM, suggests the presence of more complex transcriptional regulation. Comparatively higher StUGPase2 transcript abundance at the tuberization initiation stage as compared to the pre and post-tuberization stages indicates the prominent role of the gene during tuber development. Bioinformatics analysis of the StUGPase2 promoter region predicted the presence of various light-responsive cis-regulatory nucleotide sequences, which are considered to be associated with the light-mediated diurnal change in StUGPase2 gene expression. This research will aid in understanding the pattern of UGPase gene regulation, which is ultimately connected to potato tuber yield and bulking attributes.

UDP-glucose pyrophosphorylase: genome-wide identification, expression and functional analyses in Gossypium hirsutum.

In this study, a total of 66 UDP-glucose pyrophosphorylase (UGP) (EC 2.7.7.9) genes were identified from the genomes of four cotton species, which are the members of Pfam glycosyltransferase family (PF01702) and catalyze the reaction between glucose-1-phosphate and UTP to produce UDPG. The analysis of evolutionary relationship, gene structure, and expression provides the basis for studies on function of UGP genes in cotton. The evolutionary tree and gene structure analysis revealed that the UGP gene family is evolutionarily conserved. Collinearity and Ka/Ks analysis indicated that amplification of UGP genes is due to repetitive crosstalk generating between new family genes, while being under strong selection pressure. The analysis of cis-acting elements exhibited that UGP genes play important role in cotton growth, development, abiotic and hormonal stresses. Six UGP genes that were highly expressed in cotton fiber at 15 DPA were screened by transcriptome data and qRT-PCR analysis. The addition of low concentrations of IAA and GA3 to ovule cultures revealed that energy efficiency promoted the development of ovules and fiber clusters, and qRT-PCR showed that expression of these six UGP genes was differentially increased. These results suggest that the UGP gene may play an important role in fiber development, and provides the opportunity to plant researchers to explore the mechanisms involve in fiber development in cotton.

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.

UDP-glucose pyrophosphorylase gene affects mycelia growth and polysaccharide synthesis of Grifola frondosa

To elucidate potential roles of UDP-glucose pyrophosphorylase (UGP) in mycelial growth and polysaccharide synthesis of Grifola frondosa, a putative 2036-bp UDP-glucose pyrophosphorylase gene gfugp encoding a 53.17-kDa protein was cloned and re-annotated. Two dual promoter RNA silencing vectors of pAN7-iUGP-P-dual and pAN7-iUGP-C-dual were constructed to down-regulate gfugp expression by targeting its promoter or conserved functional sequences, respectively. Results showed that silence of gfugp promoter sequence had a higher down-regulating efficiency with slower mycelial growth and polysaccharide production than those of conserved sequence. The monosaccharide compositions/percentages of mycelial and exo-polysaccharides significantly changed with the increase of galactose and arabinose contents possibly due to block of UDP-glucose supply by gfugp silence and alteration of sugar metabolism via up-regulation of UDP-glucose-4-epimerase (gfuge) and UDP-xylose-4-epimerase (gfuxe) transcription. Our findings would provide a reference to know the biosynthesis pathway of mushroom polysaccharides and improve their production by metabolic regulation.

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom Coprinopsis cinerea.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

Detection of unapproved genetically modified potatoes in Korea using multiplex polymerase chain reaction

Several genetically modified (GM) potato cultivars with improved traits such as increased amylopectin levels, decreased asparagine levels, and reduced incidence of black spot have been developed. In this study, we describe a multiplex polymerase chain reaction (PCR) method specific for four GM potato events (EH92-527-1, AM04-1020, PH05-026-0048, and E12) that are unauthorized in Korea. The UDP-glucose pyrophosphorylase (UGPase) gene was used as the endogenous reference gene. The specificity of the primer sets was evaluated using GM potatoes and other GM crops. The limit of detection in the developed multiplex PCR was confirmed to be approximately 0.04% (w/w). Thirty-three commercial products containing potato ingredients were tested by the multiplex PCR assay, and GM potato event E12 was found in one of the 33 samples. This result suggested that the developed PCR method could be used to effectively identify unauthorized GM potatoes in Korea.

The Rice Receptor-Like Kinases DWARF AND RUNTISH SPIKELET1 and 2 Repress Cell Death and Affect Sugar Utilization during Reproductive Development.

Cell-to-cell communication precisely controls the creation of new organs during reproductive growth. However, the sensor molecules that mediate developmental signals in monocot plants are poorly understood. Here, we report that DWARF AND RUNTISH SPIKELET1 (DRUS1) and DRUS2, two closely related receptor-like kinases (RLKs), redundantly control reproductive growth and development in rice (Oryza sativa). A drus1-1 drus2 double knockout mutant, but not either single mutant, showed extreme dwarfism and barren inflorescences that harbored sterile spikelets. The gibberellin pathway was not impaired in this mutant. A phenotypic comparison of mutants expressing different amounts of DRUS1 and 2 revealed that reproductive growth requires a threshold level of DRUS1/2 proteins. DRUS1 and 2 maintain cell viability by repressing protease-mediated cell degradation and likely by affecting sugar utilization or conversion. In the later stages of anther development, survival of the endothecium requires DRUS1/2, which may stimulate expression of the UDP-glucose pyrophosphorylase gene UGP2 and starch biosynthesis in pollen. Unlike their Arabidopsis thaliana ortholog FERONIA, DRUS1 and 2 mediate a fundamental signaling process that is essential for cell survival and represents a novel biological function for the CrRLK1L RLK subfamily.

Identification of key genes involved in polysaccharide bioflocculant synthesis in Bacillus licheniformis.

The present study reports the sequenced genome of Bacillus licheniformis CGMCC 2876, which is composed of a 4,284,461 bp chromosome that contains 4,188 protein-coding genes, 72 tRNA genes, and 21 rRNA genes. Additional analysis revealed an eps gene cluster with 16 open reading frames. Conserved Domains Database analysis combined with qPCR experiments indicated that all genes in this cluster were involved in polysaccharide bioflocculant synthesis. Phosphoglucomutase and UDP-glucose pyrophosphorylase were supposed to be key enzymes in polysaccharide secretion in B. licheniformis. A biosynthesis pathway for the production of polysaccharide bioflocculant involving the integration of individual genes was proposed based on functional analysis. Overexpression of epsDEF from the eps gene cluster in B. licheniformis CGMCC 2876 increased the flocculating activity of the recombinant strain by 90% compared to the original strain. Similarly, the crude yield of polysaccharide bioflocculant was enhanced by 27.8%. Overexpression of the UDP-glucose pyrophosphorylase gene not only increased the flocculating activity by 71% but also increased bioflocculant yield by 13.3%. Independent of UDP-N-acetyl-D-mannosamine dehydrogenase gene, flocculating activity, and polysaccharide yield were negatively impacted by overexpression of the UDP-N-acetylglucosamine 2-epimerase gene. Overall, epsDEF and gtaB2 were identified as key genes for polysaccharide bioflocculant synthesis in B. licheniformis. These results will be useful for further engineering of B. licheniformis for industrial bioflocculant production. Biotechnol. Bioeng. 2017;114: 645-655. © 2016 Wiley Periodicals, Inc.

Enhanced Production of Polysaccharide Through the Overexpression of Homologous Uridine Diphosphate Glucose Pyrophosphorylase Gene in a Submerged Culture of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes).

This study aimed to improve polysaccharide production by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of the homologous UDP glucose pyrophosphorylase (UGP) gene. The effects of UGP gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production, and transcription levels of 3 genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), UGP, and α-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the UGP gene were 24.32 mg/100 mg dry weight and 1.66 g/L, respectively, which were higher by 42% and 36% than those of the wild-type strain. The transcription levels of PGM, UGP, and GLS were up-regulated by 1.6, 2.6, and 2.4-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology.

Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production.

Overexpression of UDP-glucose pyrophosphorylase from Larix gmelinii enhances vegetative growth in transgenic Arabidopsis thaliana

A UDP-glucose pyrophosphorylase gene ( LgUGPase ) was identified from Larix gmelinii, and its function in enhancing vegetative growth and cellulose biosynthesis was confirmed by analyzing transgenic Arabidopsis thaliana overexpressed LgUGPase . UDP-glucose pyrophosphorylase (UGPase), an important regulatory enzyme in carbohydrate metabolism, catalyzes the reversible production of glucose 1-phosphate and the conversion of uridine triphosphate to uridine diphosphate glucose and pyrophosphate. In this study, a larch UGPase (LgUGPase) gene was isolated from Larix gmelinii. The 1,443-bp open reading frame encodes a protein of 480 amino acids with a predicted molecular weight of 53.7 kDa and shows striking sequence similarity to UGPase proteins from Pinus taeda and Picea sitchensis. Semiquantitative reverse transcription-polymerase chain reaction showed that the LgUGPase gene was expressed primarily in the larch stem in addition to its root and leaf. Southern blot analysis indicated that LgUGPase is encoded by two genes in the L. gmelinii genome. Overexpression of LgUGPase enhanced vegetative growth in transgenic Arabidopsis and increased the contents of soluble sugars and cellulose, and thickened parenchyma cell walls. These results revealed that L. gmelinii UGPase participates in sucrose/polysaccharide metabolism and cell wall biosynthesis, suggesting that LgUGPase may be a good candidate gene for improvement of fiber cell development in plants.

Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.)

In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality.

Duplex PCR을 이용한 국내 미승인 유전자변형 감자(EH92-527-1)의 검사법 개발

A duplex polymerase chain reaction (PCR) method was developed to detect unapproved genetically modified (GM) potato (EH92-527-1) in Korea. The UDP-glucose pyrophosphorylase (UGP) gene was selected as an endogenous reference gene for potato and used to validate the specificity for 14 different crops. The primer pair EH92-F/R was designed to amplify the junction sequence between the genome and transgenic region introduced in GM potato. Its specificity was also validated using several different GM events. The detection limit of the duplex PCR method is approximately 0.05%. This duplex PCR method could be useful for monitoring cultivation of unauthorized GM potato in Korea.

Isolation and characterisation of a UDP-glucose pyrophosphorylase gene associated with fruit ripening in autumn olive (Elaeagnus umbellata Thunb.)

SummaryAutumn olive (Elaeagnus umbellata Thunb.) fruit are a rich source of many health-related nutrients. Sweetness is one of the key indicators of quality in this edible fruit. A full-length cDNA for UDP-glucose pyrophosphorylase, an enzyme involved in sugar metabolism, designated EutUGP, was cloned from autumn olive fruit. The open reading frame of EutUGP was 1,431 bp in length, and was interrupted by 18 introns. It encoded a putative polypeptide of 476 amino acids which shared high homology (85 – 87%) with other plant UGPs. The EutUGP gene was highly expressed in fruit and in young stems, but had low levels of expression in roots and leaves. Transcription of EutUGP was up-regulated and reached its maximum in Stage C (dark-yellow) fruit, then decreased slightly in Stage D (full-ripe) fruit. The highest level of accumulation of the EutUGP protein occurred at the full-ripe Stage D. The 953 bp promoter region of the EutUGP gene was isolated using PCR-based DNA-walking. Characterisation of the EutUGP gene and its promoter will contribute to our understanding of its function during ripening in autumn olive fruit.

Functional analysis of a rice late pollen-abundant UDP-glucose pyrophosphorylase (OsUgp2) promoter

OsUgp2, a rice UDP-glucose pyrophosphorylase gene, has previously been shown to preferentially express in maturing pollens and plays an important role in pollen starch accumulation. Here, a 1943 bp promoter fragment (P1943) of OsUgp2 was characterized by 5' deletion and gain-of-function experiments. P1943 and its 5' deletion derivatives (P1495, P1005, P665 and P159) were fused to GUS reporter gene and stably introduced into rice plants. Histochemical analyses of different tissues and pollens at different developmental stages of the transgenic plants showed that P1943 could only direct GUS expression in binucleate pollens. P1495 and P1005 could still drive GUS expression in binucleate pollens but at a lower level. On the other hand, neither P665 nor P159 transformant exhibited any GUS activity in pollens. Gain-of-function analyses showed that the region (-1005 to -665 relative to translation start site) combined with a minimal CaMV 35S promoter could direct GUS expression in pollens. Further analysis of 5' deletion truncated at -952, -847 and -740 delimited a 53 bp region (-1005 to -952) essential for pollen-specific expression. The 53 bp sequence contains two motifs of TTTCT and TTTC, which were known to be pollen-specific cis-elements. In addition, the same P1943-GUS fusion construct was introduced into tobacco to analyze its specificity in dicotyledon. Interestingly, the GUS expression pattern in transgenic tobacco was quite different from that in rice. High level of GUS expression was detected in mature pollens as well as leaves, roots, sepals and stigmas. These findings suggested a complicated transcriptional regulation of OsUgp2.

Cloning, Expression, and Characterization of UDP-glucose Pyrophosphorylase from Sphingomonas chungbukensis DJ77

DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in

Carbohydrate-related genes and cell wall biosynthesis in vascular tissues of loblolly pine (Pinus taeda)

Loblolly pine (Pinus taeda L.), the most widely planted tree species in the United States, is an important source of wood and wood fibers for a multitude of consumer products. Wood fibers are primarily composed of secondary cell walls, and cellulose, hemicelluloses and lignin are major components of wood. Fiber morphology and cell wall composition are important determinants of wood properties. We used comparative genomics to identify putative genes for cellulose and hemicellulose synthesis in loblolly pine that are homologous to genes implicated in cell wall synthesis in angiosperms. Sequences encoding putative secondary cell wall cellulose synthase genes, cellulose synthase-like genes, a membrane-bound endoglucanase gene, a sucrose synthase gene, a UDP-glucose pyrophosphorylase gene and GDP-mannose pyrophosphorylase genes were identified in expressed sequence tag (EST) collections from loblolly pine. Full-length coding sequences were obtained from cDNA clones isolated from a library constructed from developing xylem. Phylogenetic relationships between the genes from loblolly pine and angiosperm taxa were examined and transcriptional profiling in vascular tissues was conducted by real-time quantitative, reverse transcriptase-polymerase chain reaction. The putative cell wall synthesis genes were expressed at high levels in vascular tissues and a subset was differentially regulated in xylem and phloem tissues. Inferred phylogenetic relationships and expression patterns for the genes from loblolly pine were consistent with roles in synthesis of complex carbohydrates of the cell wall. These studies suggest functional conservation of homologous wood formation genes in gymnosperm and angiosperm taxa.