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  • 1. Ciereszko, I
    et al.
    Johansson, H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Hurry, Vaughan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kleczkowski, L.A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Phosphate status affects the gene expression, protein content and enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis.2001Ingår i: Planta, ISSN 0032-0935, Vol. 212, nr 4, s. 598-605Artikel i tidskrift (Refereegranskat)
  • 2. Ciereszko, I
    et al.
    Johansson, H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Kleczkoski, L.A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis.2001Ingår i: Biochemical Journal, Vol. 354, nr 1, s. 67-72Artikel i tidskrift (Refereegranskat)
  • 3. Ciereszko, I
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Effects of phosphate deficiency and sugars on expression of rab18 in Arabidopsis: hexokinase-dependent and okadaic acid-sensitive transduction of the sugar signal2002Ingår i: Biochimica et Biophysica Acta, Gene Structure and Expression, ISSN 0167-4781, E-ISSN 1879-2634, Vol. 1579, nr 1, s. 43-49Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The lack of phosphorus in the nutrient medium increased the expression of rab18, an abscisic acid (ABA)-responsive gene, in leaves of Arabidopsis thaliana. The expression of this gene was also upregulated after feeding the excised leaves with D-mannose and sucrose for both wild-type (wt) and aba1 (ABA-deficient) mutant plants. For aba1 mutants, both the phosphate deficiency and sugar effects on rab18 were weaker than in wt plants, suggesting possible involvement of both ABA-dependent and ABA-independent components in signalling. Transgenic Arabidopsis plants with increased hexokinase (HXK) expression had a much higher sucrose-dependent level of rab18 mRNA, implying the HXK involvement in sensing/transmitting the sugar signal. Sucrose-related induction of rab18 was completely inhibited by okadaic acid (OKA), suggesting the involvement of specific protein phosphatase(s) in transduction of the sugar signal. The results suggest that rab18 is regulated via interaction of a plethora of signals, including ABA, sugar and phosphate deficiency, and that the sugar effect is transmitted via a HXK-pathway, involving OKA-sensitive component(s). The findings prompt caution in linking the expression of rab18 solely to ABA signalling. (C) 2002 Elsevier Science B.V. All rights reserved.

  • 4. Ciereszko, I
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Glucose and mannose regulate the expression of a major sucrose synthase gene in Arabidopsis via hexokinase-dependent mechanisms2002Ingår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 40, nr 11, s. 907-911Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sucrose synthase (SuSy) is an important enzyme involved in sucrose synthesis/breakdown in all plants. Sus1, a major SuSy gene in Arabidopsis thaliana, was upregulated by sucrose, glucose and D-mannose, but not 3-O-methylglucose, when those compounds were fed to excised leaves. Mannos, was more effective than glucose or sucrose in the induction of Sus1, with strong effects observed at a concentration as low as 20, mM. When fed to the excised leaves, N-acetyl-glucosamine, an inhibitor of hexokinase (HXK) enzymatic activity, decreased sucrose- and glucose-dependent, but not mannose-dependent, upregulation of Sus1. The sucrose/glucose-dependent Sus1 expression was strongly induced in transgenic Arabidopsis HXK-overexpressing (OE) plants, whereas mannose-dependent Sus1 expression markedly decreased in OE, but not in HXK-"antisense", Arabidopsis plants. Feeding with sucrose resulted in a marked increase of glucose content in leaves, suggesting that it is glucose rather than sucrose that serves as a signal in upregulating Sus1 expression in sucrose-fed plants. The data suggest that Sus1 is regulated by a HXK-dependent pathway, with glucose and mannose effects differentially sensed/transmitted via the HXK step. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

  • 5. Ciereszko, Iwona
    et al.
    Johansson, Henrik
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Interactive effects of phosphate deficiency, sucrose and light/dark conditions on gene expression of UDP-glucose pyrophosphorylase in Arabidopsis.2005Ingår i: Journal of Plant Physiology, ISSN 0176-1617, Vol. 162, nr 3, s. 343-53Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of inorganic phosphate (Pi) status, light/dark and sucrose on expression of UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), which is involved in sucrose/ polysaccharides metabolism, were investigated using Arabidopsis wild-type (wt) plants and mutants impaired in Pi and carbohydrate status. Generally, P-deficiency resulted in increased Ugp expression and enhanced UGPase activity and protein content, as found for wt plants grown on P-deficient and complete nutrient solution, as well as for pho1 (P-deficient) mutants. Ugp was highly expressed in darkened leaves of pho1, but not wt plants, daily tight exposure enhanced Ugp expression both in wt and pho mutants. The pho1 and pho2 (Pi-accumulating) mutations had Little or no effect on leaf contents of glucose and fructose, regardless of light/dark conditions, whereas pho1 plants had much higher Levels of sucrose and starch in the dark than pho2 and wt plants. The Ugp was up-regutated when leaves were fed with sucrose in wt plants, but the expression in pho2 background was much less sensitive to sucrose supply than in wt and pho1 plants. Expression of Ugp in pgm1 and sex1 mutants (impaired in starch/sugar content) was not dependent on starch content, and not tightly correlated with soluble sugar status. Okadaic acid (OKA) effectively blocked the P-starvation and sucrose -dependent expression of Ugp in excised leaves, whereas staurosporine (STA) had only a small effect on both processes (especially in -P leaves), suggesting that P-starvation and sucrose effects on Ugp are transmitted by pathways that may share similar components with respect to their (in)sensitivity to OKA and STA. The results of this study suggest that Ugp expression is modulated by an interaction of signals derived from P-deficiency status, sucrose content and dark/ light conditions, and that light/ sucrose and P-deficiency may have additive effects on Ugp expression. (c) 2004 Elsevier GmbH. All rights reserved.

  • 6. Ciereszko, Iwona
    et al.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis2005Ingår i: Acta Physiologiae Plantarum, Vol. 27, s. 147-155Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of inorganic phosphate (Pi) deficiency on expression of genes encoding ADP-glucose pyrophosphorylase small and large subunits (ApS and ApL1, ApL2, ApL3 genes), UDP-glucose pyrophosphorylase (Ugp gene), sucrose synthase (Sus1), soluble and insoluble acid invertases (Inv and Invcw) and hexokinase (Hxk1 gene), all involved in carbohydrate metabolism, were investigated in Arabidopsis thaliana (L.) Heynh. We used soil-grown pho mutants affected in Pi status, as well as wild-type (wt) plants grown under Pi deficiency conditions in liquid medium, and leaves of wt plants fed with D-mannose. Generally, ApS, ApL1, Ugp and Inv genes were upregulated, although to a varied degree, under conditions of Pi-stress. The applied conditions had differential effects on expression of other genes studied. For instance, Sus1 was downregulated in pho1 (Pi-deficient) mutant, but was unaffected in wt plants grown in liquid medium under P-deficiency. Mannose had distinct concentration-dependent effects on expression of genes under study, possibly reflecting a dual role of mannose as a sink for Pi and as glucose analog. Feeding Pi (at up to 200 mM) to the detached leaves of wt plants strongly affected the expression of ApL1, ApL2, Sus1 and Inv genes, possibly due to an osmotic effect exerted by Pi. The data suggest that ADP-glucose and UDP-glucose pyrophosphorylases (enzymes indirectly involved in Pi recycling) as well as invertases (sucrose hydrolysis) are transcriptionally regulated by Pi-deficiency, which may play a role in homeostatic mechanisms that acclimate the plant to the Pi-stress conditions.

  • 7. Ciereszko, Iwona
    et al.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Phosphate deficiency dependent upregulation of UDP-glucose pyrophosphorylase genes is insensitive to ABA and ethylene status in Arabidopsis leaves2006Ingår i: Acta Physiologiae Plantarum, Vol. 28, s. 387-393Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of inorganic phosphate (Pi) deficiency and ABA/ethylene status on expression of UDP-glucose pyrophosphorylase (UGPase) genes (Ugp), involved in sucrose/polysaccharide metabolism, were investigated. Both wild-type (wt), aba and abi mutants (ABA-deficient and -insensitive), etr, ein and eto (ethylene resistant and overproducing) grown on Pi-deficient and complete nutrient solution, as well as pho1 (Pi-deficient) mutants of Arabidopsis thaliana were used for experiments. Generally, Pi-deficiency conditions (including mannose feeding to decrease cytosolic Pi pool) resulted in an increase of Ugp expression in the leaves, under all experimental conditions. Mutant backgrounds reflecting differences in ABA or ethylene status/ sensitivity had no effect on the level of Ugp up-regulation by Pi-stress. Furthermore, feeding ABA to the leaves of wt and pho1 plants had no effect on Ugp expression, regardless of the sucrose status in the leaves. The data suggest that Pi deficiency leading to up-regulation of Ugp acts independently of ABA and ethylene status.

  • 8.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases2017Ingår i: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 8, artikel-id 1610Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (K-m of 1.3 mM), beta-L-Ara-1-P and alpha-D-Fuc-1-P (K-m of 3.4 mM), but not beta-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (K-m of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

  • 9.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Substrate specificity and inhibitor sensitivity of plant UDP-sugar producing pyrophosphorylasesManuskript (preprint) (Övrigt vetenskapligt)
  • 10.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions2019Ingår i: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, artikel-id 1822Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.

  • 11.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Lindberg, Stina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Eriksson, Jonas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    A luminescence-based assay of UDP-sugar producing pyrophosphorylases2014Ingår i: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, nr 1, s. 57-61Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATP-sulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The activities were linear with time and proportional to the amount of the enzyme added, and were neither affected by Pi nor by DTT. For UGPase, K-m values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective K-m values with UTP, Glc-1-P and Gal-1-P were 0.4, 2.9 and 3.9 mM. Possible applications of the luminescence-based assay for not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.

  • 12.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Meng, Meng
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Gornicka, Agnieszka
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase2012Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, s. 39-45Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

  • 13.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies2017Ingår i: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, nr 6, s. 1093-1107Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

  • 14.
    Decker, Daniel
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases2018Ingår i: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 13, nr 8, artikel-id e1507406Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

  • 15. Dejardin, A
    et al.
    Sokolov, L N
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis1999Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 344, s. 503-509Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sucrose synthase (Sus) is a key enzyme of sucrose metabolism. Two Sus-encoding genes (Sus1 and Sus2) from Arabidopsis thaliana were found to be profoundly and differentially regulated in leaves exposed to environmental stresses (cold stress, drought or O-2 deficiency). Transcript levels of Sus1 increased on exposure to cold and drought, whereas Sus2 mRNA was induced specifically by O-2 deficiency. Both cold and drought exposures induced the accumulation of soluble sugars and caused a decrease in leaf osmotic potential, whereas O-2 deficiency was characterized by a nearly complete depletion in sugars. Feeding abscisic acid (ABA) to detached leaves or subjecting Arabidopsis ABA-deficient mutants to cold stress conditions had no effect on the expression profiles of Sus1 or Sus2, whereas feeding metabolizable sugars (sucrose or glucose) or non-metabolizable osmotica [poly(ethylene glycol), sorbitol or mannitol] mimicked the effects of osmotic stress on Sus1 expression in detached leaves. By using various sucrose/mannitol solutions, we demonstrated that Sus1 was up-regulated by a decrease in leaf osmotic potential rather than an increase in sucrose concentration itself. We suggest that Sus1 expression is regulated via an ABA-independent signal transduction pathway that is related to the perception of a decrease in leaf osmotic potential during stresses. In contrast, the expression of Sus2 was independent of sugar/osmoticum effects, suggesting the involvement of a signal transduction mechanism distinct from that regulating Sus1 expression. The differential stress-responsive regulation of Sus genes in leaves might represent part of a general cellular response to the allocation of carbohydrates during acclimation processes.

  • 16. Eimert, K
    et al.
    Luo, C
    Dejardin, A
    Villand, P
    Thorbjornsen, T
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves1997Ingår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 189, nr 1, s. 79-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.

  • 17. Eimert, K
    et al.
    Villand, P
    Kilian, A
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues1996Ingår i: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 170, nr 2, s. 227-232Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.

  • 18. Geisler, Matt
    et al.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Karpinski, Stanislaw
    A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis.2006Ingår i: Plant Journal, ISSN 0960-7412, Vol. 45, nr 3, s. 384-98Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Short motifs of many cis-regulatory elements (CREs) can be found in the promoters of most Arabidopsis genes, and this raises the question of how their presence can confer specific regulation. We developed a universal algorithm to test the biological significance of CREs by first identifying every Arabidopsis gene with a CRE and then statistically correlating the presence or absence of the element with the gene expression profile on multiple DNA microarrays. This algorithm was successfully verified for previously characterized abscisic acid, ethylene, sucrose and drought responsive CREs in Arabidopsis, showing that the presence of these elements indeed correlates with treatment-specific gene induction. Later, we used standard motif sampling methods to identify 128 putative motifs induced by excess light, reactive oxygen species and sucrose. Our algorithm was able to filter 20 out of 128 novel CREs which significantly correlated with gene induction by either heat, reactive oxygen species and/or sucrose. The position, orientation and sequence specificity of CREs was tested in silicio by analyzing the expression of genes with naturally occurring sequence variations. In three novel CREs the forward orientation correlated with sucrose induction and the reverse orientation with sucrose suppression. The functionality of the predicted novel CREs was experimentally confirmed using Arabidopsis cell-suspension cultures transformed with short promoter fragments or artificial promoters fused with the GUS reporter gene. Our genome-wide analysis opens up new possibilities for in silicio verification of the biological significance of newly discovered CREs, and allows for subsequent selection of such CREs for experimental studies.

  • 19. Geisler, Matt
    et al.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Karpinski, Stanislaw
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.2004Ingår i: Plant Molecular Biology, ISSN 0167-4412, Vol. 56, nr 5, s. 783-94Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

  • 20. Geisler-Lee, Jane
    et al.
    Geisler, Matt
    Coutinho, Pedro M
    Segerman, Bo
    Nishikubo, Nobuyuki
    Takahashi, Junko
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Aspeborg, Henrik
    Djerbi, Soraya
    Master, Emma
    Andersson-Gunnerås, Sara
    Sundberg, Björn
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Karpinski, Stanislaw
    Teeri, Tuula T
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Henrissat, Bernard
    Mellerowicz, Ewa J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Poplar carbohydrate-active enzymes. Gene identification and expression analyses.2006Ingår i: Plant Physiology, ISSN 0032-0889, Vol. 140, nr 3, s. 946-62Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr. & Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar (Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis (Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

  • 21. Goulas, Estelle
    et al.
    Schubert, Maria
    Kieselbach, Thomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Gardeström, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Schröder, Wolfgang
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Hurry, Vaughan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature.2006Ingår i: Plant Journal, ISSN 0960-7412, Vol. 47, nr 5, s. 720-34Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5°C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.

  • 22. Granado-Yela, C
    et al.
    García-Verdugo, C
    Carrillo, K
    Rubio DE Casas, R
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Balaguer, L
    Temporal matching among diurnal photosynthetic patterns within the crown of the evergreen sclerophyll Olea europaea L2011Ingår i: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 34, nr 5, s. 800-810Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Trees are modular organisms that adjust their within-crown morphology and physiology in response to within-crown light gradients. However, whether within-plant variation represents a strategy for optimizing light absorption has not been formally tested. We investigated the arrangement of the photosynthetic surface throughout one day and its effects on the photosynthetic process, at the most exposed and most sheltered crown layers of a wild olive tree (Olea europaea L.). Similar measurements were made for cuttings taken from this individual and grown in a greenhouse at contrasted irradiance-levels (100 and 20% full sunlight). Diurnal variations in light interception, carbon fixation and carbohydrate accumulation in sun leaves were negatively correlated with those in shade leaves under field conditions when light intensity was not limiting. Despite genetic identity, these complementary patterns were not found in plants grown in the greenhouse. The temporal disparity among crown positions derived from specialization of the photosynthetic behaviour at different functional and spatial scales: architectural structure (crown level) and carbon budget (leaf level). Our results suggest that the profitability of producing a new module may not only respond to construction costs or light availability, but also rely on its spatio-temporal integration within the productive processes at the whole-crown level.

  • 23. Igamberdiev, A U
    et al.
    Bykova, N V
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Origins and metabolism of formate in higher plants1999Ingår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 37, nr 7-8, s. 503-513Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Formate, a simple one-carbon compound, is readily metabolized in plant tissues. In greening potato tubers, similar to some procaryotes, formate is directly synthesized via a ferredoxin-dependent fixation of CO2, serving as the main precursor for carbon skeletons in biosynthetic pathways. In other plant species and tissues, formate appears as a side-product of photorespiration and of fermentation pathways, but possibly also as a product of direct CO2 reduction in chloroplasts. Formate metabolism is closely related to serine synthesis and to all subsequent reactions originating from serine. Formate may have a role in biosynthesis of numerous compounds, in energetic metabolism and in si,signal transduction pathways related to stress response. This review summarizes the current state of formate research, physiological/biochemical and molecular aspects. (C) Elsevier, Paris.

  • 24. Igamberdiev, A U
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase2000Ingår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 38, nr 10, s. 747-753Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Barley (Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)II-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate (K-i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-I in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

  • 25. Igamberdiev, A U
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments2001Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 360, s. 225-231Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3). which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg2+), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg2+] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg2+] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4 mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg2+] in chloroplasts falls to low values (0.2 mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg2+] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg2+] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATP(total)]/[ADP(total)]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg2+] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.

  • 26. Igamberdiev, A U
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Membrane potential, adenylate levels and Mg2+ are interconnected via adenylate kinase equilibrium in plant cells2003Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1607, nr 2-3, s. 111-119Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Concentrations of adenylate species and free magnesium (Mg2+) within cells are mediated by the equilibrium governed by adenylate kinase (AK), the enzyme abundant in plants in chloroplast stroma and intermembrane spaces of chloroplasts and mitochondria. Ratios of free and Mg-bound adenylates (linked to the values of [Mg2+] established under AK equilibrium) can be rationalized in terms of the overall dependence of concentrations of Mg2+ and free and Mg-bound adenylates, as well as electric potential values across the inner membranes of mitochondria and chloroplasts. The potential across the inner mitochondrial membrane, by driving adenylate translocators, equilibrates free adenylates across the inner membrane according to the Nernst equation and contributes to the ATP(total)/ADP(total) ratio in the cytosol. The ratio affects the exchange of free adenylates with chloroplasts and this, in turn, influences the value of potential across the inner chloroplast membrane. From measurements of subcellular ATP(total)/ADP(total) ratios, we suggest a method of estimating the values of potential across inner membranes of mitochondria and chloroplasts in vivo, which allows a comparison of the operation of these organelles under different physiological conditions. We discuss also how the equilibration of adenylates by AK drives adenylate transport across membranes, and establishes [Mg2+] in the cytosol and chloroplast stroma, maintaining the rates of photosynthesis and respiration. This provides a tool for metabolomic research, by which the determined concentrations of adenylate species could be used for computation of essential metabolic parameters in the cell and in subcellular compartments. (C) 2003 Elsevier B.V. All rights reserved.

  • 27. Igamberdiev, Abir U
    et al.
    Kleczkowski, Leszek
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Equilibration of adenylates in the mitochondrial intermembrane space maintains respiration and regulates cytosolic metabolism.2006Ingår i: Journal of Experimental Botany, ISSN 0022-0957, Vol. 57, nr 10, s. 2133-41Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adenylate kinase (AK) uses one each of Mg-complexed and free adenylates as substrates in both directions of its reaction. It is very active in the mitochondrial intermembrane space (IMS), but is absent from the mitochondrial matrix where low [ADP] upon intensive respiration limits the respiratory rate. AK activity in the IMS is linked to ATP/ADP exchange across the inner mitochondrial membrane by using ATP (imported from the matrix) and AMP as substrates, the latter provided by apyrase and other AMP-generating reactions. The ADP formed by AK is exported to the matrix (in exchange for ATP), providing a mechanism for regeneration of ADP during respiration. From the AK equilibrium, and taking pH values characteristic of subcellular compartments, [Mg2+] in the IMS is calculated as 0.4–0.5 mM and in the cytosol as 0.2–0.3 mM, whereas the MgATP:MgADP ratio in the IMS and cytosol is 6–9 and 10–15, respectively. These represent optimal conditions for transport of adenylates (via the maintenance of an ATPfree:ADPfree ratio close to 1) and mitochondrial respiratory rates (via the maintenance of submillimolar [ADPfree] in the IMS). This, in turn, has important consequences for mitochondrial and cytosolic metabolism, including regulation of the protein phosphorylation rate (via changes in the MgATP:AMPfree ratio) and allosteric regulation of mitochondrial and cytosolic enzymes. Metabolomic consequences are discussed in connection with the calculation of metabolic fluxes from subcompartmental distributions of total adenylates and Mg2+.

  • 28. Igamberdiev, Abir U
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Magnesium and cell energetics in plants under anoxia2011Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 437, nr 3, s. 373-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stress conditions (e.g. anoxia) frequently result in a decrease of [ATP] and in an increase of [ADP] and [AMP], with a concomitant increase of [Mg(2+)] and other cations, e.g. Ca(2+). The elevation of [Mg(2+)] is linked to the shift in the apparent equilibrium of adenylate kinase. As a result, enzymes that use Mg(2+) as a cofactor are activated, Ca(2+) activates calcium-dependent signalling pathways, and PP(i) can serve as an alternative energy source in its active form of MgPP(i) or Mg2PP(i). Under anoxic conditions in plants, an important source of PP(i) may come as a result of combined reactions of PK (pyruvate kinase) and PPDK (pyruvate, phosphate dikinase). The PP(i) formed in the PPDK/PK cycle ignites glycolysis in conditions of low [ATP] by involving PP(i)-dependent reactions. This saves ATP and makes metabolism under stress conditions more energy efficient.

  • 29.
    Igamberdiev, Abir U
    et al.
    Department of Biology, Memorial University of Newfoundland, St. John's, NL, A1B 3X9, Canada.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Metabolic systems maintain stable non-equilibrium via thermodynamic buffering2009Ingår i: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 31, nr 10, s. 1091-1099Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here, we analyze how the set of nucleotides in the cell is equilibrated and how this generates simple rules that help the cell to organize itself via maintenance of a stable non-equilibrium state. A major mechanism operating to achieve this state is thermodynamic buffering via high activities of equilibrating enzymes such as adenylate kinase. Under stable non-equilibrium, the ratios of free and Mg-bound adenylates, Mg(2+) and membrane potentials are interdependent and can be computed. The adenylate status is balanced with the levels of reduced and oxidized pyridine nucleotides through regulated uncoupling of the pyridine nucleotide pool from ATP production in mitochondria, and through oxidation of substrates non-coupled to NAD(+) reduction in peroxisomes. The set of adenylates and pyridine nucleotides constitutes a generalized cell energy status and determines rates of major metabolic fluxes. As the result, fluxes of energy and information become organized spatially and temporally, providing conditions for self-maintenance of metabolism.

  • 30. Igamberdiev, Abir U.
    et al.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium2015Ingår i: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, s. 10-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H+ and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport, and phosphate release and supply.

  • 31. Igamberdiev, Abir U
    et al.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Optimization of CO2 fixation in photosynthetic cells via thermodynamic buffering.2011Ingår i: Biosystems (Amsterdam. Print), ISSN 0303-2647, E-ISSN 1872-8324, Vol. 103, nr 2, s. 224-229Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stable operation of photosynthesis is based on the establishment of local equilibria of metabolites in the Calvin cycle. This concerns especially equilibration of stromal contents of adenylates and pyridine nucleotides and buffering of CO(2) concentration to prevent its depletion at the sites of Rubisco. Thermodynamic buffering that controls the homeostatic flux in the Calvin cycle is achieved by equilibrium enzymes such as glyceraldehyde phosphate dehydrogenase, transaldolase and transketolase. Their role is to prevent depletion of ribulose-1,5-bisphosphate, even at high [CO(2)], and to maintain conditions where the only control is exerted by the CO(2) supply. Buffering of adenylates is achieved mainly by chloroplastic adenylate kinase, whereas NADPH level is maintained by mechanisms involving alternative sinks for electrons both within the chloroplast (cyclic phosphorylation, chlororespiration, etc.) and shuttling of reductants outside chloroplast (malate valve). This results in optimization of carbon fixation in chloroplasts, illustrating the principle that the energy of light is used to support stable non-equilibrium which drives all living processes in plants.

  • 32. Igamberdiev, Abir U.
    et al.
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism2018Ingår i: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, artikel-id 318Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate serine pathway) is initiated in the cytosol and involves glycerate formation from 3phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

  • 33. Jiménez, Maria Dolores
    et al.
    Pardos, Marta
    Puértolas, Jaime
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Deep shade alters the acclimation response to moderate water streee in Quercus suber L.2009Ingår i: Forestry (London), ISSN 0015-752X, E-ISSN 1464-3626, Vol. 82, nr 3, s. 285-298Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The interactive effects of shade and drought on different morphologicaland physiological traits were addressed on Quercus suber L.seedlings. In our experiment, limited light treatment (1 percent) represented the main factor constraining cork oak seedlingsgrowth. Maximal photochemical efficiency (Fv/Fm) with light15 per cent exhibited a midday fall, but under deep shade (1per cent), Fv/Fm remained constant (<0.8 values) throughoutthe day. The quantum efficiency of photosystem II (PSII) waslower under moderate drought only in deeply shaded plants. Thedrought also interacted with light through the increase of thesoluble sugar content at 100 per cent light, but not under shade.Under deep shade, soluble sugar content tended to be even lowerunder moderate drought conditions. The chlorophyll content wasnot the highest under deep shade as could be expected, whilethe efficiency of PSII was the lowest under deep shade. We concludedthat cork oak can acclimate to moderate shade (15 per cent light),but deep shade impairs some of the physiological responses tocope with low light conditions under moderate drought stress.Plants growing under deep shade were very sensitive to moderatewater stress in terms of loss of carbon fixation capacity.

  • 34.
    Johansson, H
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Sterky, F
    Amini, B
    Lundeberg, J
    Kleczkowski, L.A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation.2002Ingår i: BBA Gene Structure and Expression, Vol. 1576, nr 1-2, s. 53-58Artikel i tidskrift (Refereegranskat)
  • 35.
    Keech, Olivier
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Gardeström, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kleczkowski, Leszek A.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Rouhier, Nicolas
    The redox control of photorespiration: from biochemical and physiological aspects to biotechnological considerations2017Ingår i: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 40, nr 4, s. 553-569Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Photorespiration is a complex and tightly regulated process occurring in photosynthetic organisms. This process can alter the cellular redox balance, notably via the production and consumption of both reducing and oxidizing equivalents. Under certain circumstances, these equivalents, as well as reactive oxygen or nitrogen species, can become prominent in subcellular compartments involved in the photorespiratory process, eventually promoting oxidative post-translational modifications of proteins. Keeping these changes under tight control should therefore be of primary importance. In order to review the current state of knowledge about the redox control of photorespiration, we primarily performed a careful description of the known and potential redox-regulated or oxidation sensitive photorespiratory proteins, and examined in more details two interesting cases: the glycerate kinase and the glycine cleavage system. When possible, the potential impact and subsequent physiological regulations associated with these changes have been discussed. In a second part, we reviewed the extent to which photorespiration contributes to cellular redox homeostasis considering, in particular, the set of peripheral enzymes associated with the canonical photorespiratory pathway. Finally, some recent biotechnological strategies to circumvent photorespiration for future growth improvements are discussed in the light of these redox regulations.

  • 36.
    Kleczkowski, L A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Back to the drawing board: Redefining starch synthesis in cereals1996Ingår i: Trends in Plant Science, ISSN 1360-1385, E-ISSN 1878-4372, Vol. 1, nr 11, s. 363-364Artikel i tidskrift (Refereegranskat)
  • 37.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    A new player in the starch field2001Ingår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 39, nr 9, s. 759-761Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A possible role of a newly discovered ADP-glucose pyrophosphatase (AGPPase) is discussed in the context of starch synthesis. The enzyme hydrolyses ADP-glucose (starch precursor) and may potentially divert the flow of carbon from starch synthase, resulting in a 'futile cycle' when 'coupled' with ADP-glucose pyrophosphorylase. The activity of AGPPase is inversely related to starch yield in sink tissues, and may be prone to inhibition by Pi and certain other products of the starch pathway. The AGPPase likely belongs to a `nudix' family of enzymes that in animal tissues and yeast are known to regulate levels of activated sugars. Some strategies for future research are underlined. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

  • 38.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    A phosphoglycerate to inorganic phosphate ratio is the major factor in controlling starch levels in chloroplasts via ADP-glucose pyrophosphorylase regulation1999Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 448, nr 1, s. 153-156Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Purified barley leaf ADP-glucose pyrophosphorylase, a key enzyme of the starch synthesis in the chloroplast stroma, was analysed with respect to its possible regulation by factors defining the metabolic/effector status of the chloroplast during light and dark conditions. The enzyme required 3-phosphoglyceric acid for the maximal activity and was inhibited by inorganic phosphate. The optimal pH for the enzyme was at circa 7.0, regardless of the presence or absence of 3-phosphoglyceric acid, whereas the maximal activation by 3-phosphoglyceric acid was observed at pH 8.5 and higher. Changes in the concentration of Mg2+ and dithiothreitol had little or no effect on the enzymatic activity of AGPase. It has been directly demonstrated for the first time that a 3-phosphoglyceric acid/inorganic phosphate ratio, a crucial regulatory parameter, could be directly related to a defined activation state of the enzyme, allowing the prediction of a relative AGPase activity under given conditions. The predicted changes in the enzyme activity were directly correlated with earlier reported responses of starch levels to the 3-phosphoglyceric acid/inorganic phosphate ratio in chloroplasts. Consequences of this for the starch biosynthesis are discussed. (C) 1999 Federation of European Biochemical Societies.

  • 39.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    GLUCOSE ACTIVATION AND METABOLISM THROUGH UDP-GLUCOSE PYROPHOSPHORYLASE IN PLANTS1994Ingår i: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 37, nr 6, s. 1507-1515Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Recent developments in studies on the characterization of properties and functions of UDP-glucose pyrophosphorylase (UGPase) in plant metabolism are presented. UGPase constitutes a reversible enzymatic step for interconversions between starch and sucrose metabolites, and is responsible for synthesis and metabolism of UDP-glucose, a major form of nucleoside diphosphoglucose in plant cells. The enzyme, although considered not to have any regulatory function, has attracted considerable interest due to its ubiquitous distribution in plants, high activity, especially in sink tissues, and because of the key role of UDP-glucose as a direct or indirect precursor of sucrose, starch and structural polysaccharides. The enzyme has been the subject of biotechnological manipulations to engineer its kinetic properties and gene expression in relation to metabolic processes at the sucrose/starch interface. Depending on tissue type, the UGPase reaction may be channelled in vivo, either toward UDP-glucose pyrophosphorolysis or synthesis, due to a metabolic coupling to other reactions of sugar pathways. Some strategies for future research on plant UGPase are discussed.

  • 40.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Is leaf ADP-glucose pyrophosphorylase an allosteric enzyme?2000Ingår i: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, ISSN 0167-4838, E-ISSN 1879-2588, Vol. 1476, nr 1, s. 103-108Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Barley leaf ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch synthesis in the chloroplast stroma, was analysed, in both directions of the reaction, with respect to details of its regulation by 3-phosphoglycerate (PGA) and inorganic phosphate (Pi) which serve as activator and inhibitor, respectively. AGPase was found to catalyse a close-to-equilibrium reaction, with the K-eq value of approximately 0.5, i.e. slightly favouring the pyrophosphorolytic direction. When the enzyme was analysed by substrate kinetics, PGA acted either as a linear (hyperbolic response) 'non-competitive' activator (forward reaction) or a linear near-'competitive' activator (reverse reaction). When the activation and inhibition patterns with PGA and Pi, respectively, were studied in detail by Dixon plots, the response curves to effecters also followed hyperbolic kinetics, with the experimentally determined K-a and K-i values on the order of micromolar. The results suggest that the regulation of AGPase proceeds via a non-cooperative mechanism, where neither of the effecters, when considered separately, induces any allosteric response. The evidence, discussed in terms of an overall kinetic mechanism/regulation of leaf AGPase, prompts caution in classifying the protein as an 'allosteric enzyme'. (C) 2000 Elsevier Science B.V. All rights reserved.

  • 41.
    Kleczkowski, Leszek A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    KINETICS AND REGULATION OF THE NAD(P)H-DEPENDENT GLYOXYLATE-SPECIFIC REDUCTASE FROM SPINACH LEAVES1995Ingår i: Zeitschrift für Naturforschung C - A Journal of Biosciences, ISSN 0939-5075, E-ISSN 1865-7125, Vol. 50, nr 1-2, s. 21-28Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Kinetic mechanism of purified spinach leaf NAD(P)H glyoxylate reductase (GR-1) was studied using either NADPH and NADH as alternative substrates with glyoxylate. The mechanism was elucidated from substrate kinetic patterns using NADH as a cofactor rather than NADPH. With NADPH varied versus glyoxylate, and with NADPH and glyoxylate varied at a constant ratio, the patterns obtained on double reciprocal plots appeared to be consistent with a ping-pong mechanism; however, kinetic patterns with NADH conclusively ruled out the ping-pong reaction in favour of the sequential addition of the reactants. Product inhibition studies with glycolate and NADP have suggested either that NADPH binds to the enzyme before glyoxylate or that the addition of substrates is a random one. Studies with active group modifiers suggested an involvement of histidine, serine and cysteine residues in GR-1 activity. Salts had little or no effect on the activity of the enzyme, with the exception of cyanide, which had an apparent K-i of ca. 2 mM. Studies with several metabolites used as possible effecters of GR-1 activity have suggested that the enzyme is modulated only by substrate availability in vivo. The apparent insensitivity of GR-1 to metabolic effecters is consistent with the proposed role of the enzyme in detoxifying glyoxylate which may act as a potent inhibitor of photosynthetic processes in plant tissues.

  • 42.
    Kleczkowski, Leszek A.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Decker, Daniel
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants2015Ingår i: Journal of Applied Glycoscience, ISSN 1344-7882, Vol. 62, nr 2, s. 25-36Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In order to serve as a glycosyl donor, a sugar or a sugar derivative (e.g. GlcA) needs to be “activated” to a highly energetic state of a nucleotide-sugar. This activation requires the involvement of specific enzymes which produce NDP-sugars (or, in one case, NMP-sugar), using NTP or NDP as substrate. The present review provides concise survey of distinct plant nucleotide-sugar pyrophosphorylases (all using NTP as one of the substrates and differing in sugar specificity) as well as nucleotide-sugar phosphorylases and sucrose synthase (all using NDP as one of substrates). The pyrophosphorylases discussed include UGPase, USPase, UAGPase, AGPase, GMPase (VTC1), and FKGP, whereas phosphorylases include ADP-Glc phosphorylase and GDP-Gal phosphorylase (VTC2/VTC5). We also discuss the activation mechanism of 3-deoxy-D-manno-octulosonic acid (Kdo) by CKS, leading to the formation of a unique NMP-linked sugar (CMP-Kdo).

  • 43.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Decker, Daniel
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation2011Ingår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 156, nr 1, s. 3-10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.

  • 44.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.2011Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, nr 3, s. 375-379Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 45.
    Kleczkowski, Leszek A.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Geisler, Matt
    Fitzek, Elisabeth
    Wilczynska, Malgorzata
    A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases2011Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 439, s. 375-379Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

  • 46.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Kunz, Sabine
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mechanisms of UDP-glucose synthesis in plants2010Ingår i: Critical reviews in plant sciences, ISSN 0735-2689, E-ISSN 1549-7836, Vol. 29, nr 4, s. 191-203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Substantial progress has been made in studies on enzymes synthesizing UDP-glucose (UDPG) which is essential for sucrose and cell wall biosynthesis, and in an array of other processes, e.g. glycosylation of proteins and lipids. The enzymes include UDPG pyrophosphorylase, UDP-sugar pyrophosphorylase (USPase) and sucrose synthase (SuSy). Genes coding for those proteins are under complex spatial and temporal regulation, and are frequently coexpressed. Recent evidence for regulation of some of the UDPG-synthesizing proteins by posttranslational modifications and oligomerization, together with discoveries of novel isozymes and unexpected locations within a cell (including chloroplasts and mitochondria) have made the studies exciting, but complex. The enzymes differ in specificity for sugar and nucleotide portions of their substrates/products, and may be involved in distinct metabolic pathways, but also in signaling. Homology models for USPase and SuSy structures are presented, based on recent crystallization of structurally related proteins. Future challenges in research on UDPG-producing enzymes are underlined.

  • 47.
    Kleczkowski, Leszek A
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sokolov, L N
    Luo, C
    Villand, P
    Molecular cloning and spatial expression of an ApL1 cDNA for the large subunit of ADP-glucose pyrophosphorylase from Arabidopsis thaliana1999Ingår i: Zeitschrift für Naturforschung C - A Journal of Biosciences, ISSN 0939-5075, E-ISSN 1865-7125, Vol. 54, nr 5-6, s. 353-358Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A cDNA, ApL1a, corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3'-ends (3'-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homologues for AGPase, and shows ca. 58-78% identity to homologous proteins from other plants/tissues. The corresponding gene was found to be expressed in all tissues examined (rosette and stem leaves, stems, flowers and fruits). The ubiquitous expression of the gene is consistent with its critical role in starch synthesis in Arabidopsis.

  • 48.
    Kleczkowski, Leszek
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Geisler, Matt
    Ciereszko, Iwona
    Johansson, Henrik
    UDP-glucose pyrophosphorylase. An old protein with new tricks.2004Ingår i: Plant Physiology, ISSN 0032-0889, Vol. 134, nr 3, s. 912-8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sugars are central to a plant's raison d'etre as products of photosynthesis. They are the ultimate source of energy and carbon skeletons for all biomolecules, and they provide the material out of which a plant builds its cell walls, fibers, and wood. Thus, regulation of any activity involved in biosynthesis of sugars, especially Suc (the major transport form of carbon in plants), is of utmost interest in understanding the growth and development strategies of a plant. Sugars are also potent regulators of gene expression, via e.g. a hexokinase (HXK) transduction mechanism that senses hexoses, or via Suc-specific or osmoticum transduction pathways, further underlying the importance of sugars in plant homeostasis.

  • 49.
    Kleczkowski, Leszek
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Martz, Francoise
    Wilczynska, Malgorzata
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Factors affecting oligomerization status of UDP-glucose pyrophosphorylase.2005Ingår i: Phytochemistry, ISSN 0031-9422, Vol. 66, nr 24, s. 2815-21Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6–9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.

  • 50. Krupa, Z
    et al.
    Siedlecka, A
    Kleczkowski, L A
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Cadmium-affected level of inorganic phosphate in rye leaves influences Rubisco subunits1999Ingår i: Acta Physiologiae Plantarum, ISSN 0137-5881, E-ISSN 1861-1664, Vol. 21, nr 3, s. 257-261Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Heavy metals, like Cd, decrease intracellular levels of essential mineral nutrient elements. Here we show the effects of the interaction between Cd and inorganic phosphate and its effects on some aspects of the photosynthetic competence of first rye leaves. The decrease in the level of small and large Rubisco subunits in the leaves of Cd-treated seedlings is discussed both in terms of the recovering effect of an additional Pi supply to the leaves, as well as of direct and indirect mechanisms of Cd-toxicity towards photosynthesis.

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