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JXB Advance Access published online on February 17, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erj084
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© The Author [2006]. 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
Received August 26, 2005
Accepted December 2, 2005

RESEARCH PAPER

Ectopic over-expression of the maize {beta}-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

Nagavalli S. Kiran 1 *, Lenka Polanská 2 *, Radka Fohlerová 1, Pavel Mazura 1, Martina Válková 3, Miloslav Smeral 4, Jan Zouhar 5, Jirí Malbeck 6, Petre I. Dobrev 6, Ivana Machácková 6, and Bretislav Brzobohaty 4 *

1 Institute of Biophysics AS CR, Královopolská 135, CZ-61265, Brno, Czech Republic; Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic
2 Institute of Experimental Botany AS CR, Rozvojová 135, CZ-16502 Prague, Czech Republic; Department of Plant Physiology, Charles University, Faculty of Science, Vinicná 5, CZ-12844 Prague 2, Czech Republic
3 Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic
4 Institute of Biophysics AS CR, Královopolská 135, CZ-61265, Brno, Czech Republic
5 Institute of Biophysics AS CR, Královopolská 135, CZ-61265, Brno, Czech Republic; Present address: Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521-0124, USA
6 Institute of Experimental Botany AS CR, Rozvojová 135, CZ-16502 Prague, Czech Republic

* To whom correspondence should be addressed.
Bretislav Brzobohaty, E-mail: brzoboha{at}ibp.cz


  Abstract

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize {beta}-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent Km more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

Keywords: {beta}-glucosidase; chloroplast; cytokinin metabolism; ectopic expression; hormone conjugation; zeatin-O-glucoside.

*These authors contributed equally to the work presented here.


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