biologia plantarum

International journal on Plant Life established by Bohumil Nìmec in 1959

Biologia plantarum 51:673-682, 2007 | DOI: 10.1007/s10535-007-0141-6

Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae

J. Frébortová1,2,*, P. Galuszka3, T. Werner4, T. Schmülling4, I. Frébort3
1 Laboratory of Growth Regulators, Faculty of Science, Palacký University, Olomouc, Czech Republic
2 Institute of Experimental Botany of the Academy of Science, Olomouc, Czech Republic
3 Department of Biochemistry, Faculty of Science, Palacký University, Olomouc, Czech Republic
4 Institute of Biology and Applied Genetics, Free University of Berlin, Berlin, Germany

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 µkat g-1, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.

Keywords: cytokinin metabolism; cytokinin oxidase
Subjects: Arabidopsis thaliana; cytochrome c oxidase; cytokinin oxidase/dehydrogenase; cytokinins; Saccharomyces cerevisiae

Received: October 13, 2006; Accepted: May 17, 2007; Published: December 1, 2007  Show citation

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Frébortová, J., Galuszka, P., Werner, T., Schmülling, T., & Frébort, I. (2007). Functional expression and purification of cytokinin dehydrogenase from Arabidopsis thaliana (AtCKX2) in Saccharomyces cerevisiae. Biologia plantarum, 51(4), 673-682. doi: 10.1007/s10535-007-0141-6
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    References

    1. Armstrong, D.J., Firtel, R.A.: Cytokinin oxidase activity in the cellular slime mold Dictyostelium discoideum.-Dev. Biol. 136: 491-499, 1989. Go to original source...
    2. Bechet, J., Greenson, M., Wiame, J.M.: Mutations affecting the repressibility of arginine biosynthetic enzymes in Saccharomyces cerevisiae.-Eur. J. Biochem. 12: 31-39, 1970. Go to original source...
    3. Bilyeu, K.D., Cole, J.L., Laskey, J.G., Riekhof, W.R., Esparza, T.J., Kramer, M.D., Morris, R.O.: Molecular and biochemical characterization of a cytokinin oxidase from maize.-Plant Physiol. 125: 378-386, 2001. Go to original source...
    4. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.-Anal. Biochem. 72: 248-254, 1976. Go to original source...
    5. Brownlee, B.G., Hall, R.H., Whitty, C.D.: 3-Methyl-2-butenal: an enzymatic degradation product of the cytokinin N 6-(Delta;2-isopentenyl)adenine.-Can. J. Biochem. 53: 37-41, 1975. Go to original source...
    6. Frébort, I., ©ebela, M., Galuszka, P., Werner, T., Schmülling, T., Peè, P.: Cytokinin oxidase/dehydrogenase assay: optimized procedures and applications-Anal. Biochem. 306: 1-7, 2001. Go to original source...
    7. Frébortová, J., Fraaije, M.W., Galuszka, P., ©ebela, M., Peè, P., Hrbáè, J., Bilyeu, K.D., English, J.T., Frébort, I.: Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.-Biochem. J. 380: 121-130, 2004. Go to original source...
    8. Galuszka, P., Frébort, I. ©ebela, M., Peè, P.: Degradation of cytokinins by cytokinin oxidases in plants.-Plant Growth Regul. 32: 315-327, 2000. Go to original source...
    9. Galuszka, P., Frébort, I., ©ebela, M., Sauer, P., Jacobsen, S., Peè, P.: Cytokinin oxidase or dehydrogenase? Mechanism of the cytokinin degradation in plants.-Eur. J. Biochem. 268: 450-461, 2001. Go to original source...
    10. Galuszka, P., Frébortová, J., Werner, T., Yamada, M., Strnad, M., Schmülling, T., Frébort, I.: Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression.-Eur. J. Biochem. 271: 3990-4002, 2004. Go to original source...
    11. Galuszka, P., Popelková, H., Werner, T., Frébortová, J., Pospí¹ilová, H., Mik, V., Köllmer, I., Schmülling, T., Frébort, I.: Biochemical characterization of cytokinin oxidases/dehydrogenases from Arabidopsis thaliana expressed in Nicotiana tabacum L.-J. Plant Growth Regul. in press, 2007. Go to original source...
    12. Gerhäuser, D., Bopp, M.: Cytokinin oxidases in mosses. 1 Metabolism of kinetin and benzyladenine in vivo.-J. Plant Physiol. 135: 680-685, 1990. Go to original source...
    13. Gietz, R.D., Sugino, A.: New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.-Gene 74: 527-534, 1988. Go to original source...
    14. Gietz, R.D., Woods, R.A.: High-efficiency transformation of plasmid DNA into yeast.-Methods mol. Biol. 177: 85-97, 2001.
    15. Houba-Herin, N., Pethe, C., D'Alayer, J., Laloue, M.: Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts.-Plant J. 17: 615-626, 1999. Go to original source...
    16. Jones, R.J., Schreiber, B.M.N., McNeil, K., Brenner, M.L., Foxon, G.: Cytokinin levels and oxidase activity during maize kernel development.-In: Kamínek, M., Mok, D.W.S., Za¾ímalová, E. (ed.): Physiology and Biochemistry of Cytokinins in Plants. Pp. 235-239. SPB Academic Publishing, The Hague 1992.
    17. Jones, R.J., Schreiber, B.M.N.: Role and function of cytokinin oxidase in plants.-Plant Growth Regul. 23: 123-134, 1997. Go to original source...
    18. Kopeèný, D., Pethe, C., ©ebela, M., Houba-Herin, N., Madzak, C., Majira, A., Laloue, M.: High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica.-Biochimie 87: 1011-1022, 2005. Go to original source...
    19. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.-Nature 227: 680-685, 1970. Go to original source...
    20. Libreros-Minotta C.A., Tipton P.A.: A colorimetric assay for cytokinin oxidase.-Anal. Biochem. 231: 339-341, 1995. Go to original source...
    21. Malito, E., Coda, A., Bilyeu, K.D., Fraaije, M.W., Mattevi, A.: Structures of Michaelis and product complexes of plant cytokinin dehydrogenase: implication for flavoenzyme catalysis.-J. mol. Biol. 341: 1237-1249, 2004. Go to original source...
    22. Massonneau, A., Houba-Herin, N., Pethe, C., Madzak, C., Falque, M., Mercy, M., Kopeèný, D., Majira, A., Laloue, M.: Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs.-J. exp. Bot. 55: 2549-2557, 2004. Go to original source...
    23. Mok, D.W., Mok, M.C.: Cytokinin metabolism and action.-Annu. Rev. Plant Physiol. Plant mol. Biol. 52: 89-118, 2001. Go to original source...
    24. Morris, R.O., Bilyeu, K.D., Laskey, J.G., Cheikh, N.N.: Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.-Biochem. Biophys. Res. Commun. 255: 328-333, 1999. Go to original source...
    25. Paèes, V., Werstiuk, E., Hall, R.H.: Conversion of N 6-(Delta;2-isopentenyl)adenosine to adenosine by enzyme activity in tobacco tissue.-Plant Physiol. 48: 775-778, 1971. Go to original source...
    26. Popelková, H., Fraaije, M.W., Novák, O., Frébortová, J., Bilyeu, K.D., Frébort, I.: Kinetic and chemical analyses of the cytokinin oxidase/dehydrogenase catalysed reaction: correlations with the crystal structure.-Biochem. J. 398: 113-124, 2006. Go to original source...
    27. Popelková, H., Galuszka, P., Frébortová, J., Bilyeu, K.D., Frébort, I.: Cytokinin dehydrogenase: characterization and structure homology modeling of the flavoprotein catabolizing plant hormones cytokinins.-In: Pandalai, S.G. (ed.): Recent Research Developments in Proteins. Vol. 2. Pp. 63-81. Transworld Research Network, Kerala 2004.
    28. Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S., Frommer, W.B.: NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis:-FEBS Lett. 370: 264-268, 1995. Go to original source...
    29. Sakakibara, H.: Cytokinins: activity, biosynthesis, and translocation.-Annu. Rev. Plant Biol. 57: 431-449, 2006. Go to original source...
    30. Schmülling, T., Werner, T., Riefler, M., Krupková, E., Bartrina y Manns, I.: Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species.-J. Plant Res. 116: 241-252, 2003. Go to original source...
    31. Thompson, J.R., Register, E., Curotto, J., Kurtz, M., Kelly, R.: An improved protocol for the preparation of yeast cells for transformation by electroporation.-Yeast 14: 565-571, 1998. Go to original source...
    32. Van Kast, C.A., Laten, H.: Cytokinin utilization by adenine requiring mutants of the yeast Saccharomyces cerevisiae.-Plant Physiol. 83: 726-727, 1987. Go to original source...
    33. Wang, J., Letham, D.S.: Cytokinin oxidase-purification by affinity chromatography and activation by caffeic acid.-Plant Sci. 112: 161-166, 1995. Go to original source...
    34. Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H.: Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.-Plant Cell 15: 1-20, 2003. Go to original source...
    35. Werner, T., Motyka, V., Strnad, M., Schmülling, T.: Regulation of plant growth by cytokinin.-Proc. nat. Acad. Sci. USA 98: 10487-10492, 2001. Go to original source...
    36. Zang, S.H., Yu, H, Goh, C.J.: Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid.-Plant mol. Biol. 51: 237-248, 2003.

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