caa a22 4500 463171829 CHVBK 20180406164815.0 cr unu---uuuuu 170326e20070901xx s 000 0 eng 10.1007/s00344-007-9008-5 doi (NATIONALLICENCE)springer-10.1007/s00344-007-9008-5 Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from Arabidopsis thaliana Expressed in Nicotiana tabacum L [Elektronische Daten] [Petr Galuszka, 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 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. Springer Science+Business Media, LLC, 2007 Activity staining nationallicence Arabidopsis thaliana nationallicence Cytokinin oxidase/dehydrogenase nationallicence pH optimum nationallicence Substrate specificity nationallicence Nicotiana tabacum nationallicence Galuszka Petr Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Popelková Hana Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Werner Tomáš Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut Frébortová Jitka Laboratory of Growth Regulators, Palacký University/Institute of Experimental Botany of the Academy of Science, Czech Republic, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Pospíšilová Hana Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Mik Václav Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Köllmer Ireen Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut Schmülling Thomas Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut Frébort Ivo Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Journal of Plant Growth Regulation Springer-Verlag 26/3(2007-09-01), 255-267 0721-7595 26:3<255 2007 26 344 https://doi.org/10.1007/s00344-007-9008-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00344-007-9008-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Galuszka Petr Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Popelková Hana Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Werner Tomáš Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut NATIONALLICENCE 700 1- Frébortová Jitka Laboratory of Growth Regulators, Palacký University/Institute of Experimental Botany of the Academy of Science, Czech Republic, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Pospíšilová Hana Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Mik Václav Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Köllmer Ireen Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut NATIONALLICENCE 700 1- Schmülling Thomas Institute of Biology/Applied Genetics, Free University of Berlin, Albrecht-Thaer-Weg 6, D−14195, Berlin, Germany aut NATIONALLICENCE 700 1- Frébort Ivo Division of Molecular Biology, Department of Biochemistry, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 773 0- Journal of Plant Growth Regulation Springer-Verlag 26/3(2007-09-01), 255-267 0721-7595 26:3<255 2007 26 344 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer