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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 Galuszka
  • Hana Popelková
  • Tomáš Werner
  • Jitka Frébortová
  • Hana Pospíšilová
  • Václav Mik
  • Ireen Köllmer
  • Thomas Schmülling
  • Ivo Frébort
Article

Abstract

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) 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.

Keywords

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

Notes

Acknowledgments

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.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Petr Galuszka
    • 1
  • 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

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