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Differential tissue/organ-dependent expression of two sucrose- and cold-responsive genes for UDP-glucose pyrophosphorylase in Populus.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
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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.

Place, publisher, year, edition, pages
2007. Vol. 389, no 2, 186-95 p.
Keyword [en]
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
URN: urn:nbn:se:umu:diva-12827DOI: doi:10.1016/j.gene.2006.11.006PubMedID: 17196771OAI: diva2:152498
Available from: 2008-05-27 Created: 2008-05-27 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, MengKleczkowski, Leszek A
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