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Characterisation of the ATP-Dependent Phosphofructokinase Gene Family from Arabidopsis Thaliana

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Characterisation of the ATP-Dependent Phosphofructokinase Gene Family from Arabidopsis Thaliana View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 2401–2410 Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana Angelika Mustropha,1, Uwe Sonnewaldb, Sophia Biemeltb,* a Humboldt-University Berlin, Institute of Biology, AG Plant Physiology, Philippstrasse 13, 10115 Berlin, Germany b Friedrich-Alexander University Erlangen-Nuremberg, Department of Biochemistry, Staudtstrasse 5, 91058 Erlangen, Germany Received 1 March 2007; revised 15 April 2007; accepted 16 April 2007 Available online 30 April 2007 Edited by Mark Stitt addition to the widely distributed ATP-dependent phospho- Abstract Plants possess two different types of phosphofructo- kinases, an ATP-dependent (PFK) and a pyrophosphate-depen- fructokinase (PFK, EC 2.7.1.11), in plants and a number of dent form (PFP). While plant PFPs have been investigated in prokaryotes a second type of phosphofructokinase is present detail, cDNA clones coding for PFK have not been identified using pyrophosphate (PPi) instead of ATP as phosphoryl in Arabidopsis thaliana. Searching the A. thaliana genome re- donor (pyrophosphate-fructose-6-phosphate-phosphotransfer- vealed 11 putative members of a phosphofructokinase gene fam- ase, PFP, EC 2.7.1.90). Phosphorylation of Fru6P catalyzed by ily. Among those, four sequences showed high homology to the PFK is virtually irreversible in vivo, while PFP reacts near alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted equilibrium and catalyzes the reaction in both directions. In in elevated PFK, but not PFP activity after transient expression addition a third, ADP-dependent phosphofructokinase has in tobacco leaves suggesting that they encode Arabidopsis PFKs. been reported which seems to be specific for certain groups RT-PCR revealed different tissue-specific expression of the indi- of archaebacteria [3]. vidual forms. Furthermore, analysis of GFP fusion proteins indi- cated their presence in different sub-cellular compartments. Since its discovery in 1980s, plant PFP has been character- Ó 2007 Federation of European Biochemical Societies. Published ized in detail in a range of plant species at the biochemical by Elsevier B.V. All rights reserved. and molecular level (for example, Solanum tuberosum [4]; Ricinus communis [5]; Citrus · paradisii [6]; Saccharum officina- Keywords: Phosphofructokinase; Agrobacterium infiltration; rum [7]). Additionally to plants, the enzyme was also found in Arabidopsis thaliana several microorganisms, for example in Amycolatopsis methan- olica [8] and Entamoeba histolytica [9]. PFP generally consists of two different subunits, alpha and beta, which usually form a heterotetramer (e.g. [10,11]). The enzyme activity is highly regulated by fructose-2,6-bisphosphate (Fru2,6BP) [1,12],in 1. Introduction contrast to animals, where PFK is regulated by Fru2,6BP [13]. Even though PFP is highly regulated, its function is not Glycolysis is a central metabolic pathway which is present in clearly resolved. Transgenic potato plants with more than all organisms. It provides substrates to fuel energy production 90% reduction in PFP activity did not provide evidence for a and anabolic processes of living cells. Glycolysis is also of role of PFP in controlling the rate of glycolysis [14]. However, importance for adaptations to stress conditions such as nutri- PFP might be involved in adaptation of plants to non-optimal ent limitations, cold, drought or oxygen deficiency. The regu- conditions, such as phosphate deficiency or low oxygen stress lation of the glycolytic process is essential for all cell types. [15–17]. Key regulatory enzymes in plant glycolysis are pyruvate kinase While PFP has been studied in detail, plant PFK has re- and phosphofructokinase [1]. ceived only little attention. Besides extensive studies of Musa Phosphofructokinase catalyzes the interconversion of fruc- cavendishii PFKs [18–22], the enzyme was analysed in Daucus tose-6-phosphate (Fru6P) and fructose-1.6-bisphosphate. carota, Cucumis sativus, Solanum tuberosum, Ricinus commu- Whereas most glycolytic enzymes are remarkably conserved nis, and Spinacia oleracea [23–32] (see Table 1 for details). Nei- between different organisms, various types of phosphofructo- ther subunit composition nor the size of the protein was similar kinases exist with a very complex evolutionary history [2].In in all plant species tested. One major problem in PFK isolation was the instability of the enzyme during purification [22,27]. However, as a consistent result, cytosolic and plastidic iso- forms were found in many plant species (Table 1), as known *Corresponding author. Fax: +49 9131 8528254. for several glycolytic enzymes [1]. Very recently, a PFK was E-mail address: [email protected] (S. Biemelt). isolated from spinach leaves, and sequenced [31]. This is the 1Present address: Department of Botany and Plant Sciences, Center for first and only report of a plant PFK protein sequence up to Plant Cell Biology, University of California, Riverside, CA 92521, now. USA. However, while this work was started, neither the nucleotide sequence nor the amino acid composition of any plant PFK Abbreviations: Fru2,6BP, fructose-2,6-bisphosphate; G3PDH, was known. The importance of this enzyme for regulation of glycerol-3-phosphate-dehydrogenase; PFK, ATP-dependent phospho- fructokinase; PFP, pyrophosphate-fructose-6-phosphate-phospho- metabolism prompted us to search for PFK-encoding se- transferase; PPi, pyrophosphate; TPI, triosephosphate-isomerase quences in plants. Therefore we have chosen the model plant 0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.060 2402 A. Mustroph et al. / FEBS Letters 581 (2007) 2401–2410 Table 1 Overview of approaches to purify ATP-dependent PFKs from different plant species Plant species Organ Characteristics of the purified protein(s) Reference Cytosolic form Plastidic form Size of the Presence and/or Size of the Presence and/or protein MW size (kDa) of SU protein MW size (kDa) of SU (kDa) (kDa) Cucumis sativus Seed 180 40, 42, 47 180 39, 41.5, 53 [23] Daucus carota Root 5000 60, 68 [24] Glycine max Nodules 200, 2000 33, 55, 59, 62 [25] Lycopersicon esculentum Fruit 180 35 [26] Musa acuminata Fruit 210 Unstable 160 Unstable [22] Ricinus communis Seed Present Unstable 220 57 [27] Ricinus communis Seed Unknown 61.5 [28] Solanum tuberosum Tuber 200–800 46.3, 49.5, 50, 53 [29] Spinacia oleracea Leaf Unknown 50–70 [30] Spinacia oleracea Leaf 200 52 [31] Triticum aestivum Seed 182 [32] Data were taken from the references indicated. MW, molecular weight; SU, subunit composition. Arabidopsis thaliana. The gene coding for PFK in Anabaena sion-DNA-polymerase (Finnzymes, Finland; PFK2, 5, 6). The result- (strain PCC7120, Gene Entry: all7335) was used to find homo- ing PCR products were sub-cloned into the following vectors: PFK1, logues genes in the Arabidopsis genome. Thereby seven Ara- 3, 4: pCR 2.1 (Invitrogen, Karlsruhe, Germany), PFK7: pCR Blunt (Invitrogen); PFK2, 5, 6: pGEMT (Promega). bidopsis genes were identified which were essentially the The amplified products were sequenced to verify the sequence iden- same as previously predicted by phylogenetic analysis [31,33]. tity. The corresponding cDNAs were cloned and tested for their The fragments were excised using the Asp 718 and BamHI (PFK1–4, PFP- and PFK activity by transiently over-expressing them 6, 7) or Asp718 and XbaI restriction sites (PFK5) and inserted into a Bin19-derived binary vector [35] between the CaMV 35 S promoter in tobacco leaves. Thereby we could experimentally prove that and octopin synthase polyadenylation signal. these seven open reading frames code for PFK in A. thaliana. For localisation studies GFP was C-terminally fused to fragments In addition, tissue specific expression and sub-cellular localiza- encoding putative PFKs as well as to the proposed signal peptide of tion of the PFK isoforms were analysed. PFK4 (PFK4-SP). To this end, the cDNA clones were excised without the stop codon using the Asp 718 and SalI restriction sites of PFK1–4 and the Asp 718 and SpeI restrictions sites of PFK5–7, respectively. The fragments were then cloned into the pBinGFP vector [36] which 2. Materials and methods had been digested accordingly. The binary constructs were transformed into Agrobacterium tum- 2.1. Plant material and growth conditions efaciens, strain CV58C1, carrying the virulence plasmid pGV2260 Both Nicotiana tabacum (cv. Samsun NN) and Nicotiana benthami- [37], which were used for transient expression of the genes in tobacco ana were grown in a greenhouse with 16 h supplemental light (ca. plants as described below. 250 lmol quanta PAR À2 sÀ1) followed by 8 h darkness. The tempera- ture regime followed the day/night cycle with 25 °C/18 °C. Arabidopsis 2.4. Agrobacterium mediated transient gene expression in tobacco plants plants (cv. Columbia) were grown in a growth chamber under 9-h-illu- For transient transformation, the method described by Bendahmane mination at 22 °C. et al. [38] was used. Briefly, Agrobacterium tumefaciens cells harbour- ing the putative PFK cDNAs were grown overnight in 50 ml YEB 2.2. Sequence alignments medium with kanamycin, rifampycin, ampicillin, 1 mM MES pH 5.6 PFK and PFP protein sequences were obtained from public dat- and 20 lM acetosyringone. Cells were harvested by centrifugation at abases (http://www.ncbi.nlm.nih.gov/entrez, http://www.arabidopsis. 3500 · g and 4 °C for 20 min. The pellet was washed with sterile water org/). Putative phosphofructokinase sequences from Oryza sativa and to remove excess of the medium. After a second centrifugation step, Populus trichocarpa were obtained by BLAST search of the respective the pellet was re-suspended in infiltration buffer containing 10 mM databases with the Arabidopsis protein sequences (http://www.tigr.org/ MgCl2, 10 mM MES pH 5.6 and 0.1 mM acetosyringone, and adjusted tdb/e2k1/osa1/, http://genome.jgi-psf.org/Poptr1_1/). Sequence align- to a final concentration of OD600 of 1.0.
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