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

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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.

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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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Author notes

  1. Hana Popelková

    Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Michigan, Ann Arbor, 48109-1048, USA

Authors and Affiliations

  1. Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Petr Galuszka, Hana Popelková, Hana Pospíšilová, Václav Mik & Ivo Frébort

  2. Laboratory of Growth Regulators, Palacký University/Institute of Experimental Botany of the Academy of Science, Czech Republic, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Jitka Frébortová

  3. Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany

    Tomáš Werner, Ireen Köllmer & Thomas Schmülling

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  1. Petr Galuszka
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Correspondence to Petr Galuszka.

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Galuszka, P., Popelková, H., Werner, T. et al. Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from Arabidopsis thaliana Expressed in Nicotiana tabacum L.. J Plant Growth Regul 26, 255–267 (2007). https://doi.org/10.1007/s00344-007-9008-5

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