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JXB Advance Access published online on May 12, 2009
Journal of Experimental Botany, doi:10.1093/jxb/erp126
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© The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize

Mária Smehilová1,*, Petr Galuszka1, Kristin D. Bilyeu2, Pavel Jaworek1, Marta Kowalska1, Marek Sebela1, Michaela Sedlárová3, James T. English4 and Ivo Frébort1

1Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
2United States Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, 110 Waters Hall, Columbia, MO 65211, USA
3Department of Botany, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
4Division of Plant Sciences, University of Missouri, 110 Waters Hall, Columbia, MO 65211 USA

* To whom correspondence should be addressed. E-mail: maria.smehilova{at}upol.cz

Cytokinin dehydrogenase (CKX; EC 1.5.99.12 [EC] ) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in plant cells. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the subcellular localization of ZmCKX10, and compare its biochemical properties with those of ZmCKX1. Expression profiling of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine their transcript abundance and organ-specific expression. For determination of the subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, the ZmCKX1–GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10–GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1–GFP formed significantly more mass in comparison with the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor and trans-zeatin, N6-({Delta}2-isopentenyl)adenine (iP) and N6-({Delta}2-isopentenyl)adenosine (iPR) as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates.

Key words: Apoplast, CKX, CKX activity assay, cytokinin, cytosol, GFP, Pichia pastoris expression system, subcellular localization, tomato hairy root transformation, Zea mays

Received 18 February 2009; Revised 20 March 2009 Accepted 28 March 2009


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