Journal of Plant Growth Regulation

, Volume 26, Issue 3, pp 255–267 | Cite as

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from Arabidopsis thaliana Expressed in Nicotiana tabacum L.

  • Petr GaluszkaEmail author
  • Hana Popelková
  • Tomáš Werner
  • Jitka Frébortová
  • Hana Pospíšilová
  • Václav Mik
  • Ireen Köllmer
  • Thomas Schmülling
  • Ivo Frébort


Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N 6 -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N 6 -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.


Activity staining Arabidopsis thaliana Cytokinin oxidase/dehydrogenase pH optimum Substrate specificity Nicotiana tabacum 



This work was supported by the grants 522/06/0703 from the Grant Agency, Czech Republic (PG), MSM 6198959216 from the Ministry of Education, Youth and Physical Education, Czech Republic (IF), and DFG grant Schm 814/17-2, Germany (TS). The authors thank Lenka Luhová for technical assistance with the histochemical localization.


  1. Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ, Kramer MD, Morris RO (2001) Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:378–386PubMedCrossRefGoogle Scholar
  2. 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 72:248–254PubMedCrossRefGoogle Scholar
  3. Brzobohatý B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, Palme K (1993) Release of active cytokinin by a beta-glucosidase localized to the maize root meristem. Science 262:1051–1054PubMedCrossRefGoogle Scholar
  4. Burch LR, Stuchbury T (1986) Purification and properties of adenosine nucleosidases from tomato (Lycopersicon esculentum) roots and leaves. J Plant Physiol 125:267–273Google Scholar
  5. Chen CM, Kristopeit SM (1981) Deribosylation of cytokinin ribonucleosides by adenosine nucleosidase from wheat germ cells. Plant Physiol 68:1020–1023PubMedCrossRefGoogle Scholar
  6. Frébort I, Šebela M, Galuszka P, Werner T, Schmülling T, Peč P (2002) Cytokinin oxidase/cytokinin dehydrogenase assay: Optimized procedures and applications. Anal Biochem 306:1–7PubMedCrossRefGoogle Scholar
  7. Frébortová J, Fraaije MW, Galuszka P, Šebela M, Peč P, Hrbáč J, Novák O, Bilyeu KD, English JT, Frébort I (2004) Catalytic reaction of cytokinin dehydrogenase: Preference for quinones as electron acceptors. Biochem J 380:121–130PubMedCrossRefGoogle Scholar
  8. Frébortová J, Galuszka P, Popelková H, Lenobel R, Šebela M, Werner T, Schmülling T, Frébort I (2007) Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae. Biol Plant (in press)Google Scholar
  9. Fusseder A, Ziegler P (1988) Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum. Planta 173:104–109CrossRefGoogle Scholar
  10. Galuszka P, Frébort I, Šebela M, Sauer P, Jacobsen S, Peč P (2001) Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur J Biochem 268:450–461PubMedCrossRefGoogle Scholar
  11. Galuszka P, Frébortová J, Werner T, Yamada M, Strnad M, Schmülling T, Frébort I (2004) Cytokinin oxidase/dehydrogenase genes in barley and wheat: cloning and heterologous expression. Eur J Biochem 271: 3990–4002PubMedCrossRefGoogle Scholar
  12. Galuszka P, Frébortová J, Luhová L, Bilyeu KD, English JT, Frébort I (2005) Tissue localization of cytokinin dehydrogenase in maize: Possible involvement of quinone species generated from plant phenolics by other enzymatic systems in the catalytic reaction. Plant Cell Physiol 46:716–728PubMedCrossRefGoogle Scholar
  13. Gatz C, Frohberg C, Wendenburg R (1992) Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenig tobacco plants. Plant J 2:397–404PubMedGoogle Scholar
  14. Guranowski A, Schneider Z (1977) Purification and characterization of adenosine nucleosidase from barley leaves. Biochim Biophys Acta 482:145–158PubMedGoogle Scholar
  15. Haidoune M, Pethe C, Laloue M, Mornet R (1994) Transformations of the natural cytokinins zeatin in aqueous acidic media. J Chem Soc Perkin Trans 1:3009–3012CrossRefGoogle Scholar
  16. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  17. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  18. Hirose N, Makita N, Yamaya T, Sakakibara H (2005) Functional characterization and expression analysis of a gene, OsENT2, encoding an equilibrative nucleoside transporter in rice suggest a function in cytokinin transport. Plant Physiol 138:196–206PubMedCrossRefGoogle Scholar
  19. Houba-Hérin N, Pethe C, d’Alayer J, Laloue M (1999) Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626PubMedCrossRefGoogle Scholar
  20. Kakimoto T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases. Plant Cell Physiol 42:677–685PubMedCrossRefGoogle Scholar
  21. Kopečný D, Pethe C, Šebela M, Houba-Hérin N, Madzak C, Majira A, Laloue M (2005) High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica. Biochimie 87:1011–1022PubMedCrossRefGoogle Scholar
  22. Laloue M, Fox JE (1989) Cytokinin oxidase from wheat: Partial purification and general properties. Plant Physiol 90:899–906PubMedGoogle Scholar
  23. Laskey GL, Patterson P, Bilyeu KD, Morris RO (2003) Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor. Plant Growth Regul 40:189–196CrossRefGoogle Scholar
  24. Letham DS, Palni LMS, Tao G-Q, Gollnow BI, Bates CM (1983) Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays. J Plant Growth Regul 2:103–115CrossRefGoogle Scholar
  25. Malito E, Coda A, Bilyeu KD, Fraaije MW, Mattevi A (2004) Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implications for flavoenzyme catalysis. J Mol Biol 341:1237–1249PubMedCrossRefGoogle Scholar
  26. Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, Mercy M, Kopečný D, Majira A, Rogowsky P, Laloue M (2004) Maize cytokinin oxidase genes: Differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557PubMedCrossRefGoogle Scholar
  27. Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118PubMedCrossRefGoogle Scholar
  28. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333PubMedCrossRefGoogle Scholar
  29. Motyka V, Vaňková R, Čapková V, Petrášek J, Kamínek M, Schmülling T (2003) Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol Plant 117:11–21CrossRefGoogle Scholar
  30. Popelková H, Galuszka P, Frébortová J, Bilyeu KD, Frébort I (2004) Cytokinin dehydrogenase: Characterization and structure homology modelling of the flavoprotein catabolizing plant hormones cytokinins. In: Pandalai SG (ed), Recent Research Developments in Proteins, vol. 2. Kerala, India: Transworld Research Network, pp 63–81Google Scholar
  31. Popelková H, Fraaije MW, Novák O. Frébortová J, Bilyeu KD, Frébort I (2006) Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure. Biochem J 398:113–124PubMedCrossRefGoogle Scholar
  32. Sakano Y, Okada Y, Matsunaga A, Suwama T, Kaneko T, Ito K, Noguchi H, Abe I (2004) Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.). Phytochemistry 65:2439–2446PubMedCrossRefGoogle Scholar
  33. Sakakibara H (2006) Cytokinins: Activity, biosynthesis, and translocation. Annu Rev Plant Biol 57:431–439PubMedCrossRefGoogle Scholar
  34. Spíchal L, Rakova NY, Riefler M., Mizuno T, Romanov GA, Strnad M, Schmülling T (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45:1299–1305PubMedCrossRefGoogle Scholar
  35. Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci U S A 98:10487–10492PubMedCrossRefGoogle Scholar
  36. Werner T, Motyka V, Laucou V, Smets R, van Onckelen H, Schmülling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550PubMedCrossRefGoogle Scholar
  37. Werner T, Köllmer I, Bartrina I, Holst K, Schmülling T (2006) New insights into the biology of cytokinin degradation. Plant Biol 8:371–381PubMedCrossRefGoogle Scholar
  38. Wesenberg GE, Phillips GN, Han BW, Bitto E, Bingman CA, Bae E (2005) X-ray structure of cytokinin oxidase/dehydrogenase (CKX) from Arabidopsis thaliana AT5G21482. PDB entry 2EXR. Available at
  39. Yang SH, Yu H, Goh CJ (2002) Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter. J Exp Bot 53:1899–1907PubMedCrossRefGoogle Scholar
  40. Yang SH, Yu H, Goh CJ (2003) Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Petr Galuszka
    • 1
    Email author
  • Hana Popelková
    • 1
    • 4
  • Tomáš Werner
    • 3
  • Jitka Frébortová
    • 2
  • Hana Pospíšilová
    • 1
  • Václav Mik
    • 1
  • Ireen Köllmer
    • 3
  • Thomas Schmülling
    • 3
  • Ivo Frébort
    • 1
  1. 1.Division of Molecular Biology, Department of BiochemistryPalacký UniversityOlomoucCzech Republic
  2. 2.Laboratory of Growth Regulators, Palacký University/Institute of Experimental Botany of the Academy of Science, Czech RepublicOlomoucCzech Republic
  3. 3.Institute of Biology/Applied Genetics, Free University of BerlinBerlinGermany
  4. 4.Department of MolecularCellular and Developmental Biology, University of MichiganMichiganUSA

Personalised recommendations