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Plant UDP-glucose Pyrophosphorylase: Function and Regulation
Umeå University, Faculty of Science and Technology, Plant Physiology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase.

In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants.

In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins.

We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Place, publisher, year, edition, pages
Umeå: Fysiologisk botanik , 2008. , 38 p.
Keyword [en]
UDP-glucose pyrophosphorylase, carbohydrate metabolism, in vivo regulation, T-DNA knockout, heterologous expression, isozyme, mutagenesis, kinetic property, oligomerization
National Category
Botany
Identifiers
URN: urn:nbn:se:umu:diva-1796ISBN: 978-91-7264-604-9 (print)OAI: oai:DiVA.org:umu-1796DiVA: diva2:141997
Public defence
2008-09-12, KB3B1, KBC, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2008-09-01 Created: 2008-09-01 Last updated: 2009-06-25Bibliographically approved
List of papers
1. Differential tissue/organ-dependent expression of two sucrose- and cold-responsive genes for UDP-glucose pyrophosphorylase in Populus.
Open this publication in new window or tab >>Differential tissue/organ-dependent expression of two sucrose- and cold-responsive genes for UDP-glucose pyrophosphorylase in Populus.
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2007 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 389, no 2, 186-95 p.Article in journal (Refereed) Published
Abstract [en]

Plant UDP-glucose (UDPG) pyrophosphorylase (UGPase) is involved in the production/metabolism of UDPG, a key metabolite for sucrose and cell wall biosynthesis. Two highly similar cDNAs (UGP1 and UGP2) corresponding to UGPase were isolated from cDNA libraries of hybrid aspen (Populus tremula x tremuloides). Expression of both UGPs, as studied by DNA microarrays and EST abundance, was compared to that of three sucrose synthase genes (SUS1–3), also involved in UDPG synthesis. Generally, the UGPs had lower expression than SUS1 and SUS2 genes (especially in tension wood and cambium), with the notable exception of leaves, primary roots and flowers. Based on real-time quantitative PCR, UGP1 in root xylem, leaves and male flowers was by far the predominant transcript, while in other tissues both UGP1 and UGP2 had comparable expression. In leaves, the UGP1 gene, but not UGP2, was upregulated by light and short-term sucrose feeding. Cold treatment led to dramatic organ-specific changes in relative expression of both genes, with UGP2 being upregulated either transiently (leaves), long-term (stems) or not at all (roots), whereas UGP1 was cold-upregulated in all organs. Individual or overall UGP expression patterns only weakly correlated with UGPase activity/protein; however, UGPase activity and protein were correlated in all tissues/conditions. The data suggest that UGPs are differentially expressed at the tissue level and in response to metabolic feedback (sucrose) and cold stress, and point to a tight posttranscriptional/translational control and, possibly, distinct roles for those genes.

Keyword
Cold, DNA; Complementary, Flowers/genetics/metabolism, Gene Expression Regulation; Plant, Genes; Plant, Glucosyltransferases/genetics/metabolism, Phylogeny, Plant Leaves/genetics/metabolism, Plant Proteins/genetics/metabolism, Populus/*enzymology/genetics/metabolism, Protein-Serine-Threonine Kinases/genetics/metabolism, Sucrose/*metabolism, UTP-Glucose-1-Phosphate Uridylyltransferase/*genetics/metabolism, Xylem/genetics/metabolism
Identifiers
urn:nbn:se:umu:diva-12827 (URN)doi:10.1016/j.gene.2006.11.006 (DOI)17196771 (PubMedID)
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2017-12-14Bibliographically approved
2. UDP-glucose pyrophosphorylase is not rate limiting, but is essential in arabidopsis
Open this publication in new window or tab >>UDP-glucose pyrophosphorylase is not rate limiting, but is essential in arabidopsis
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2009 (English)In: Plant and Cell Physiology, ISSN 0032-0781, E-ISSN 1471-9053, Vol. 50, no 5, 998-1011 p.Article in journal (Refereed) Published
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) produces UDP-glucose which is essential for sucrose and polysaccharide synthesis. Using Arabidopsis, we demonstrated that two UGPase genes (UGP1 and UGP2) are differentially expressed in a variety of organs, with UGP1 being pre-dominant. Co-expression analyses of UGP genes suggest that UGP1 is closely co-regulated with carbohydrate metabolism genes, late embryogenesis and seed loading, while UGP2 is co-regulated with stress response genes, fertilized flowers and photosynthetic genes. We have used Arabidopsis mutants for the UGP genes to characterize the role of both genes. The UGPase activity/protein was reduced by 70, 10 and 85% in ugp1, ugp2 and ugp1/ugp2 double mutant (DK) plants, respectively. A decrease in UGPase activity/protein was accompanied by an increase in expression of USP, a gene for UDP-sugar pyrophos-phorylase, suggesting a compensatory mechanism. Generally, the mutants had no effects on soluble sugar/starch content (except in certain cases for DK plants), and there were no differences in cell wall composition/content between the wild type and the mutants. On the other hand, DK plants had greater hypocotyl and root lengths. When grown in the field, the mutants had as much as a 50% decrease in the number of seeds produced (consistent with a substantial decrease in field fitness), suggesting that they would be outcompeted in the field in a few generations. Overall, the data suggest that UGPase is not rate limiting for sucrose/starch and cell wall synthesis, but that it is essential in Arabidopsis.

Place, publisher, year, edition, pages
Kyoto: Japanese society of plant physiologists, 2009
Keyword
Callose, Cellulose, Cell wall synthesis, Seed fitness, Sucrose synthesis
Identifiers
urn:nbn:se:umu:diva-23573 (URN)10.1093/pcp/pcp052 (DOI)
Available from: 2009-06-25 Created: 2009-06-25 Last updated: 2017-12-13Bibliographically approved
3. Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.
Open this publication in new window or tab >>Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.
2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 6, 967-972 p.Article in journal (Refereed) Published
Abstract [en]

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10–30% lower than that of UGPase-2, depending on direction of the reaction, whereas its Km values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03–0.14 mM vs. 0.07–0.36 mM, respectively). Both proteins were “true” UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.

Identifiers
urn:nbn:se:umu:diva-9502 (URN)doi:10.1016/j.bbapap.2008.02.021 (DOI)18395530 (PubMedID)
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2017-12-14Bibliographically approved
4. Domain-specific determinants of catalysis/ substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase
Open this publication in new window or tab >>Domain-specific determinants of catalysis/ substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase
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2009 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1794, 1734-1742 p.Article in journal (Refereed) Published
Abstract [en]

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K(m) with PPi (up to 33mM), whereas Ncut deletions had greatly increased K(m) with UDPG (up to 57mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K(m) values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

Identifiers
urn:nbn:se:umu:diva-23574 (URN)10.1016/j.bbapap.2009.08.009 (DOI)
Available from: 2009-06-25 Created: 2009-06-25 Last updated: 2017-12-13Bibliographically approved

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