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The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

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Abstract

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−3H]trans-zeatin ([2−3H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−3H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−3H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.

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Acknowledgments

This work was supported by the Grant Agency of the Czech Republic (206/03/0313). The authors thank Dr. Miroslav Kamínek for critical reading of manuscript and Vanda Lacmanová and Marie Korecká for excellent technical assistance.

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Authors and Affiliations

  1. Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, Prague 6, CZ-16502, Czech Republic

    Alena Gaudinová, Petre I. Dobrev, Blanka Šolcová & Václav Motyka

  2. Laboratory of Growth Regulators, Palacký University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Šlechtitelů, Olomouc, CZ-78371, Czech Republic

    Ondřej Novák & Miroslav Strnad

  3. Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital, I. P. Pavlova 6, Olomouc, CZ-77520, Czech Republic

    David Friedecký

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  1. Alena Gaudinová
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  2. Petre I. Dobrev
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  3. Blanka Šolcová
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  4. Ondřej Novák
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  7. Václav Motyka
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Correspondence to Václav Motyka.

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Gaudinová, A., Dobrev, P.I., Šolcová, B. et al. The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves. J Plant Growth Regul 24, 188–200 (2005). https://doi.org/10.1007/s00344-005-0043-9

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  • DOI: https://doi.org/10.1007/s00344-005-0043-9

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