, Volume 224, Issue 1, pp 108–124 | Cite as

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

  • Sonia Dorion
  • Daniel P. Matton
  • Jean RivoalEmail author
Original Article


A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6×His-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.


Isoform Meristem NTP metabolism Nucleoside diphosphate kinase Root metabolism Solanum tuberosum 



Isopropyl β-D-thiogalactoside




Glyceraldehyde 3-phosphate dehydrogenase


2-[N-morpholino]ethanesulphonic acid


Nucleoside diphosphate kinase


Ni-nitrilotriacetic acid




Phosphoenolpyruvate carboxylase


Insoluble polyvinylpolypyrrolidone


Tobacco etch virus



This work was supported by a Discovery Grant from NSERC to J. R. S. D. was the recipient of a Postdoctoral Fellowship from NSERC. We thank Dr. W.C. Plaxton (Queen’s University) for the gift of affinity purified anti-cytosolic aldolase IgG. We are grateful to Dr. D. Morse and Y. Wang (Université de Montréal) for help with PsbA detection. Propur Inc. (Saint-Ambroise, QC, Canada) provided Russet Burbank seed potatoes used in this study and their help is gratefully acknowledged.


  1. Agou F, Raveh S, Veron M (2000) The binding mode of human nucleoside diphosphate kinase B to single- strand DNA. J Bioenerg Biomembr 32:285–292CrossRefPubMedGoogle Scholar
  2. Albrecht G, Mustroph A (2003) Localization of sucrose synthase in wheat roots: increased in situ activity of sucrose synthase correlates with cell wall thickening by cellulose deposition under hypoxia. Planta 217:252–260PubMedGoogle Scholar
  3. Behal RH, Oliver DJ (1997) Biochemical and molecular characterization of fumarase from plants: purification and characterization of the enzyme, cloning, sequencing, and expression of the gene. Arch Biochem Biophys 348:65–74CrossRefPubMedGoogle Scholar
  4. Bernard MA, Ray NB, Olcott MC, Hendricks SP, Mathews CK (2000) Metabolic functions of microbial nucleoside diphosphate kinases. J Bioenerg Biomembr 32:259–267CrossRefPubMedGoogle Scholar
  5. Berry EA, Huang LS, DeRose VJ (1991) Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria. J Biol Chem 266:9064–9077PubMedGoogle Scholar
  6. Bidel LP, Renault P, Pages L, Rivière LM (2000) Mapping meristem respiration of Prunus persica (L.) Batsch seedlings: potential respiration of the meristems, O2 diffusional constraints and combined effects on root growth. J Exp Bot 51:755–768CrossRefPubMedGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Choi G, Yi H, Lee J, Kwon YK, Soh MS, Shin B, Luka Z, Hahn TR, Song PS (1999) Phytochrome signalling is mediated through nucleoside diphosphate kinase 2. Nature 401:610–613CrossRefPubMedGoogle Scholar
  9. Contento AL, Kim SJ, Bassham DC (2004) Transcriptome profiling of the response of Arabidopsis suspension culture cells to sucrose starvation. Plant Physiol 135:2330–2347CrossRefPubMedGoogle Scholar
  10. Dancer J, Neuhaus HE, Stitt M (1990a) Subcellular compartmentation of uridine nucleotides and nuceloside-5′-diphosphate kinase in leaves. Plant Physiol 92:637–641Google Scholar
  11. Dancer J, Veith R, Feil R, Komor E, Stitt M (1990b) Independent changes of inorganic pyrophosphate and the ATP/ADP or UTP/UDP ratios in plant cell suspension cultures. Plant Sci 66:59–63CrossRefGoogle Scholar
  12. De Leonardis S, De Lorenzo G, Borraccino G, Dipierro S (1995) A specific ascorbate free radical reductase isozyme participates in the regeneration of ascorbate for scavenging toxic oxygen species in potato tuber mitochondria. Plant Physiol 109:847–851PubMedGoogle Scholar
  13. van Dongen JT, Roeb GW, Dautzenberg M, Froehlich A, Vigeolas H, Minchin PE, Geigenberger P (2004) Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds. Plant Physiol 135:1809–1821CrossRefPubMedGoogle Scholar
  14. Dorion S, Rivoal J (2003) Quantification of uridine 5′-diphosphate (UDP)-glucose by high-performance liquid chromatography and its application to nonradioactive assay for nucleoside diphosphate kinase using UDP-glucose pyrophosphorylase as a coupling enzyme. Anal Biochem 323:188–196CrossRefPubMedGoogle Scholar
  15. Dorion S, Parveen, Jeukens J, Matton DP, Rivoal J (2005) Cloning and characterization of a cytosolic isoform of triosephosphate isomerase developmentally regulated in potato leaves. Plant Sci 168:183–194CrossRefGoogle Scholar
  16. Ellis KJ, Morrison JF (1982) Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87:405–426PubMedGoogle Scholar
  17. Escobar Galvis ML, Hakansson G, Alexciev K, Knorpp C (1999) Cloning and characterisation of a pea mitochondrial NDPK. Biochimie 81:1089–1096CrossRefPubMedGoogle Scholar
  18. Escobar Galvis ML, Marttila S, Hakansson G, Forsberg J, Knorpp C (2001) Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein. Plant Physiol 126:69–77CrossRefPubMedGoogle Scholar
  19. Farré EM, Tiessen A, Roessner U, Geigenberger P, Trethewey RN, Willmitzer L (2001) Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiol 127:685–700PubMedGoogle Scholar
  20. Finan PM, White IR, Redpath SH, Findlay JB, Millner PA (1994) Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum. Plant Mol Biol 25:59–67CrossRefPubMedGoogle Scholar
  21. Freije JM, Blay P, MacDonald NJ, Manrow RE, Steeg PS (1997) Site-directed mutation of NM23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro. J Biol Chem 272:5525–5532CrossRefPubMedGoogle Scholar
  22. Gout E, Boisson AM, Aubert S, Douce R, Bligny R (2001) Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. carbon-13 and phosphorous-31 nuclear magnetic resonance studies. Plant Physiol 125:912–925CrossRefPubMedGoogle Scholar
  23. Hajirezaei M, Sonnewald U, Viola R, Carlisle SM, Dennis DT, Stitt M (1994) Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta 192:16–30Google Scholar
  24. Hertzberg M, Aspeborg H, Schrader J, Andersson A, Erlandsson R, Blomqvist K, Bhalerao R, Uhlen M, Teeri TT, Lundeberg J, Sundberg B, Nilsson P, Sandberg G (2001) A transcriptional roadmap to wood formation. Proc Natl Acad Sci USA 98:14732–14737CrossRefPubMedGoogle Scholar
  25. Inoue H, Takahashi M, Oomori A, Sekiguchi M, Yoshioka T (1996) A novel function for nucleoside diphosphate kinase in Drosophila. Biochem Biophys Res Commun 218:887–892CrossRefPubMedGoogle Scholar
  26. Johansson M, Mackenzie-Hose A, Andersson I, Knorpp C (2004) Structure and mutational analysis of a plant mitochondrial nucleoside diphosphate kinase. Identification of residues involved in serine phosphorylation and oligomerization. Plant Physiol 136:3034–3042CrossRefPubMedGoogle Scholar
  27. Kimura N, Shimada N, Fukuda M, Ishijima Y, Miyazaki H, Ishii A, Takagi Y, Ishikawa N (2000) Regulation of cellular functions by nucleoside diphosphate kinases in mammals. J Bioenerg Biomembr 32:309–315CrossRefPubMedGoogle Scholar
  28. Knorpp C, Johansson M, Baird AM (2003) Plant mitochondrial nucleoside diphosphate kinase is attached to the membrane through interaction with the adenine nucleotide translocator. FEBS Lett 555:363–366CrossRefPubMedGoogle Scholar
  29. Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol 127:1694–1710CrossRefPubMedGoogle Scholar
  30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  31. Lambeth DO, Mehus JG, Ivey MA, Milavetz BI (1997) Characterization and cloning of a nucleoside-diphosphate kinase targeted to matrix of mitochondria in pigeon. J Biol Chem 272:24604–24611CrossRefPubMedGoogle Scholar
  32. Lantin S, O’Brien M, Matton DP (1999) Pollination, wounding and jasmonate treatments induce the expression of a developmentally regulated pistil dioxygenase at a distance, in the ovary, in the wild potato Solanum chacoense Bitt. Plant Mol Biol 41:371–386CrossRefPubMedGoogle Scholar
  33. Lascu I, Gonin P (2000) The catalytic mechanism of nucleoside diphosphate kinases. J Bioenerg Biomembr 32:237–246CrossRefPubMedGoogle Scholar
  34. Lombardi D, Mileo AM (2003) Protein interactions provide new insight into Nm23/nucleoside diphosphate kinase functions. J Bioenerg Biomembr 35:67–71CrossRefPubMedGoogle Scholar
  35. Lubeck J, Soll J (1995) Nucleoside diphosphate kinase from pea chloroplasts: purification, cDNA cloning and import into chloroplasts. Planta 196:668–673CrossRefPubMedGoogle Scholar
  36. Mancuso S, Boselli M (2002) Characterisation of the oxygen fluxes in the division, elongation and mature zones of Vitis roots: influence of oxygen availability. Planta 214:767–774CrossRefPubMedGoogle Scholar
  37. Mills WR, Joy KW (1980) A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves. Planta 148:75–83CrossRefGoogle Scholar
  38. Moon H, Lee B, Choi G, Shin D, Prasad DT, Lee O, Kwak SS, Kim DH, Nam J, Bahk J, Hong JC, Lee SY, Cho MJ, Lim CO, Yun DJ (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc Natl Acad Sci USA 100:358–363CrossRefPubMedGoogle Scholar
  39. Nomura T, Fukui T, Ichikawa A (1991) Purification and characterization of nucleoside diphosphate kinase from spinach leaves. Biochim Biophys Acta 1077:47–55PubMedGoogle Scholar
  40. Nomura T, Yatsunami K, Honda A, Sugimoto Y, Fukui T, Zhang J, Yamamoto J, Ichikawa A (1992) The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence. Arch Biochem Biophys 297:42–45CrossRefPubMedGoogle Scholar
  41. Novikova GV, Moshkov IE, Smith AR, Hall MA (2003) Nucleoside diphosphate kinase is a possible component of the ethylene signal transduction pathway. Biochemistry (Mosc) 68:1342–1348CrossRefGoogle Scholar
  42. Pan L, Kawai M, Yano A, Uchimiya H (2000) Nucleoside diphosphate kinase required for coleoptile elongation in rice. Plant Physiol 122:447–452CrossRefPubMedGoogle Scholar
  43. Parks REJ, Agarwal RP (1973) Nucleoside diphosphokinases. Enzymes 8:307–334Google Scholar
  44. Reiter WD, Vanzin GF (2001) Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol Biol 47:95–113CrossRefPubMedGoogle Scholar
  45. Riou-Khamlichi C, Menges M, Healy JM, Murray JA (2000) Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Mol Cell Biol 20:4513–4521CrossRefPubMedGoogle Scholar
  46. Rivoal J, Ricard B, Pradet A (1989) Glycolytic and fermentative enzyme induction during anaerobiosis in rice seedlings. Plant Physiol Biochem 27:43–52Google Scholar
  47. Rivoal J, Dunford R, Plaxton WC, Turpin DH (1996) Purification and properties of four phosphoenolpyruvate carboxylase isoforms from the green alga Selenastrum minutum: evidence that association of the 102-kDa catalytic subunit with unrelated polypeptides may modify the physical and kinetic properties of the enzyme. Arch Biochem Biophys 332:47–57CrossRefPubMedGoogle Scholar
  48. Rivoal J, Plaxton WC, Turpin DH (1998) Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii. Kinetic, structural and immunological evidence that the green algal enzyme is distinct from the prokaryotic and higher plant enzymes. Biochem J 331:201–209PubMedGoogle Scholar
  49. Rivoal J, Trzos S, Gage DA, Plaxton WC, Turpin DH (2001) Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum. J Biol Chem 276:12588–12597CrossRefPubMedGoogle Scholar
  50. Roberts JK (1990) Observation of uridine triphosphate:glucose-1-phosphate uridylyltransferase activity in maize root tips by saturation transfer 31P-NMR. Estimation of cytoplasmic PPi. Biochim Biophys Acta 1051:29–36CrossRefPubMedGoogle Scholar
  51. Roberts JK, Callis J, Wemmer D, Walbot V, Jardetzky O (1984a) Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proc Natl Acad Sci USA 81:3379–3383CrossRefGoogle Scholar
  52. Roberts JKM, Callis J, Jardetzky O, Walbot V, Freeling M (1984b) Cytoplasmic acidosis as a determinant of flooding intolerance in plants. Proc Natl Acad Sci USA 81:6029–6033CrossRefGoogle Scholar
  53. Roberts JKM, Aubert S, Gout E, Bligny R, Douce R (1997) Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance. Plant Physiol 113:191–199PubMedGoogle Scholar
  54. Rolletschek H, Weber H, Borisjuk L (2003) Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes. Plant Physiol 132:1196–1206CrossRefPubMedGoogle Scholar
  55. Rontein D, Dieuaide-Noubhani M, Dufourc EJ, Raymond P, Rolin D (2002) The metabolic architecture of plant cells. Stability of central metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells. J Biol Chem 277:43948–43960CrossRefPubMedGoogle Scholar
  56. Roscher A, Emsley L, Raymond P, Roby C (1998) Unidirectional steady state rates of central metabolism enzymes measured simultaneously in a living plant tissue. J Biol Chem 273:25053–25061CrossRefPubMedGoogle Scholar
  57. Rothbauer U, Hofmann S, Muhlenbein N, Paschen SA, Gerbitz KD, Neupert W, Brunner M, Bauer MF (2001) Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria. J Biol Chem 276:37327–37334CrossRefPubMedGoogle Scholar
  58. Roymans D, Willems R, Van Blockstaele DR, Slegers H (2002) Nucleoside diphosphate kinase (NDPK/NM23) and the waltz with multiple partners: possible consequences in tumor metastasis. Clin Exp Metastasis 19:465–476CrossRefPubMedGoogle Scholar
  59. Schrader J, Nilsson J, Mellerowicz E, Berglund A, Nilsson P, Hertzberg M, Sandberg G (2004) A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity. Plant Cell 16:2278–2292CrossRefPubMedGoogle Scholar
  60. Seifert GJ (2004) Nucleotide sugar interconversions and cell wall biosynthesis: how to bring the inside to the outside. Curr Opin Plant Biol 7:277–284CrossRefPubMedGoogle Scholar
  61. Seitz B, Klos C, Wurm M, Tenhaken R (2000) Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ-specific expression patterns. Plant J 21:537–546CrossRefPubMedGoogle Scholar
  62. Shen Y, Kim JI, Song PS (2005) NDPK2 as a signal transducer in the phytochrome-mediated light signalling. J Biol Chem 280:5740–5749CrossRefPubMedGoogle Scholar
  63. Shin DH, In JG, Lim YP, Hasunuma K, Choi KS (2004) Molecular cloning and characterization of nucleoside diphosphate (NDP) kinases from Chinese cabbage (Brassica campestris). Mol Cells 17:86–94PubMedGoogle Scholar
  64. Smith CR, Knowles VL, Plaxton WC (2000) Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation. Eur J Biochem 267:4477–4485CrossRefPubMedGoogle Scholar
  65. Sommer D, Song PS (1994) A plant nucleoside diphosphate kinase homologous to the human Nm23 gene product: purification and characterization. Biochim Biophys Acta 1222:464–470CrossRefPubMedGoogle Scholar
  66. Sorscher SM, Steeg P, Feramisco JR, Buckmaster C, Boss GR, Meinkoth J (1993) Microinjection of an nm23 specific antibody inhibits cell division in rat embryo fibroblasts. Biochem Biophys Res Commun 195:336–345CrossRefPubMedGoogle Scholar
  67. Spetea C, Hundal T, Lundin B, Heddad M, Adamska I, Andersson B (2004) Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen. Proc Natl Acad Sci USA 101:1409–1414CrossRefPubMedGoogle Scholar
  68. St-Pierre B, Vazquez-Flota FA, De Luca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11:887–900CrossRefPubMedGoogle Scholar
  69. Sweetlove LJ, Mowday B, Hebestreit HF, Leaver CJ, Millar AH (2001) Nucleoside diphosphate kinase III is localized to the inter-membrane space in plant mitochondria. FEBS Lett 508:272–276CrossRefPubMedGoogle Scholar
  70. Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K (1998) Phytochrome-mediated light signals are transduced to nucleoside diphosphate kinase in Pisum sativum L. cv. Alaska. J Photochem Photobiol B 45:113–121CrossRefPubMedGoogle Scholar
  71. Timmons L, Shearn A (2000) Role of AWD/nucleoside diphosphate kinase in Drosophila development. J Bioenerg Biomembr 32:293–300CrossRefPubMedGoogle Scholar
  72. Turner WL, Plaxton WC (2000) Purification and characterization of cytosolic pyruvate kinase from banana fruit. Biochem J 352:875–882CrossRefPubMedGoogle Scholar
  73. Yang LM, Lamppa GK (1996) Rapid purification of a chloroplast nucleoside diphosphate kinase using CoA-affinity chromatography. Biochim Biophys Acta 1294:99–102PubMedGoogle Scholar
  74. Yano A, Umeda M, Uchimiya H (1995) Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.). Plant Mol Biol 27:1053–1058CrossRefPubMedGoogle Scholar
  75. Zhang J, Fukui T, Ichikawa A (1995) A third type of nucleoside diphosphate kinase from spinach leaves: purification, characterization and amino-acid sequence. Biochim Biophys Acta 1248:19–26PubMedGoogle Scholar
  76. Zimmermann S, Baumann A, Jaekel K, Marbach I, Engelberg D, Frohnmeyer H (1999) UV-responsive genes of Arabidopsis revealed by similarity to the Gcn4- mediated UV response in yeast. J Biol Chem 274:17017–17024CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Institut de Recherche en Biologie VégétaleUniversité de MontréalMontréalCanada

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