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New cytokinin metabolites in IPT transgenic Arabidopsis thaliana plants

Authors

  • Tomáš Werner,

    1. Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 783 71 Olomouc, Czech Republic
    2. Institute of Biology, Free University of Berlin, D-14195 Berlin, Germany
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  • Jan Hanuš,

    1. Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 142 20 Praha, Czech Republic
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  • Jan Holub,

    1. Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 783 71 Olomouc, Czech Republic
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  • Thomas Schmülling,

    1. Laboratory for Plant Biochemistry and Physiology, Department of Biology, University of Antwerp, B-2610 Antwerp, Belgium
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  • Harry Van Onckelen,

    1. Laboratory for Plant Biochemistry and Physiology, Department of Biology, University of Antwerp, B-2610 Antwerp, Belgium
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  • Miroslav Strnad

    Corresponding author
    1. Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 783 71 Olomouc, Czech Republic
      * Corresponding author, e-mail: strnad@aix.upol.cz
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  • Edited by D. Van Der Straeten

* Corresponding author, e-mail: strnad@aix.upol.cz

Abstract

Two novel cytokinin metabolites were identified in transgenic Arabidopsis thaliana (L.) Heynh. plants containing the bacterial IPT gene under the transcriptional control of a heat-regulated promoter. After cyclic heat-shock treatment, the endogenous cytokinin concentrations were elevated up to 100-fold compared to the wild-type plants. More then 20 different cytokinin metabolites were found, with zeatin-type cytokinins being the most abundant. The metabolic inactivation of these compounds occurred predominantly through N-glucosylation. No significant accumulation of isopentenyladenine-type cytokinins, and only a small increase in dihydrozeatin metabolites, was observed. Subsequent studies of the abundant, unidentified conjugates revealed the presence of zeatin and dihydrozeatin diglucoside conjugates. Structural analysis, utilizing electrospray-liquid tandem mass spectrometry, identified these as a zeatin-O-glucoside-9-glucoside and dihydrozeatin-O-glucoside-9-glucoside, respectively. A third unknown metabolite, was tentatively identified as a phosphorylated form of zeatin-9-glucoside. The biological activity of these compounds in three cytokinin bioassays was low. A comparison of the cytokinin pattern in transgenic and wild-type plants indicates that these specific metabolites accumulate as a consequence of enhanced cytokinin biosynthesis, and are probably involved in the homeostatic mechanisms that control endogenous cytokinin levels.

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