Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts

Authors

  • Nicole Houba-Hérin,

    1. 1 Laboratoire de Biologie Cellulaire INRA, Route de S t-Cyr, 78026 Versailles Cedex, France, and2Laboratoire de Microséquençage des Protéines, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France
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  • 1 , Claude Pethe,

    1. 1 Laboratoire de Biologie Cellulaire INRA, Route de S t-Cyr, 78026 Versailles Cedex, France, and2Laboratoire de Microséquençage des Protéines, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France
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  • 1 , Jacques D’Alayer,

    1. 1 Laboratoire de Biologie Cellulaire INRA, Route de S t-Cyr, 78026 Versailles Cedex, France, and2Laboratoire de Microséquençage des Protéines, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France
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  • and 2 Michel Laloue 1 ,

    1. 1 Laboratoire de Biologie Cellulaire INRA, Route de S t-Cyr, 78026 Versailles Cedex, France, and2Laboratoire de Microséquençage des Protéines, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France
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  • EMBL Data Library accession number Y18377.

*For correspondence (fax +33 130833099; e-mail laloue@versailles.inra.fr ).
†These authors contributed equally to the work presented here.

Summary

Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of theN6-(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea ([3H]-azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme. Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD-dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.

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