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UDP-glucose pyrophosphorylase is not rate limiting, but is essential in arabidopsis
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Department of Plant Biology, Southern Illinois University.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Department of Plant Physiology, University of Copenhagen.
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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. Vol. 50, no 5, 998-1011 p.
Keyword [en]
Callose, Cellulose, Cell wall synthesis, Seed fitness, Sucrose synthesis
URN: urn:nbn:se:umu:diva-23573DOI: 10.1093/pcp/pcp052OAI: diva2:225249
Available from: 2009-06-25 Created: 2009-06-25 Last updated: 2015-04-29Bibliographically approved
In thesis
1. Plant UDP-glucose Pyrophosphorylase: Function and Regulation
Open this publication in new window or tab >>Plant UDP-glucose Pyrophosphorylase: Function and Regulation
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.
UDP-glucose pyrophosphorylase, carbohydrate metabolism, in vivo regulation, T-DNA knockout, heterologous expression, isozyme, mutagenesis, kinetic property, oligomerization
National Category
urn:nbn:se:umu:diva-1796 (URN)978-91-7264-604-9 (ISBN)
Public defence
2008-09-12, KB3B1, KBC, Umeå, 10:00 (English)
Available from: 2008-09-01 Created: 2008-09-01 Last updated: 2009-06-25Bibliographically approved

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Meng, MengJohansson, HenrikKleczkowski, Leszek A.
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