CHANGES IN CYTOKININ CONTENT AND CYTOKININ OXIDASE ACTIVITY IN RESPONSE TO DEREPRESSION OF
ipt
GENE TRANSCRIPTION IN TRANSGENIC TOBACCO CALLI AND PLANTS
Václav Motyka
1
, Martin Faiss
2
, Miroslav Strnad
3
, Miroslav Kamínek
4
, Thomas Schmülling
2
Plant Physiol 112: 1035-1043.
1
Institute of Experimental Botany, Ke Dvoru 15, CZ-16630 Prague 6, Czech Republic
2
Universität Tübingen, Lehrstuhl fⁿr Allgemeine Genetik, Auf der Morgenstelle 28, D-72076
Tübingen, Germany
3
Institute of Experimental Botany, Sokolovskß 6, CZ-77200 Olomouc, Czech Republic
4
De Montfort University Norman Borlaug Centre for Plant Science, Institute of Experimental
Botany, Ke dvoru 15, CZ-16630 Prague 6, Czech Republic
ABSTRACT
Metabolic control of cytokinin oxidase by its substrate was investigated
in planta
using wild-type (WT) and conditionally ipt gene expressing transgenic (IPT) tobacco (
Nicotiana tabacum
L.) callus cultures and plants. The derepression of the tetracycline(Tc)-dependent
ipt
gene transcription was followed by a progressive more than hundredfold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 to 20 h after gene derepression and was after 13 days 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity as a consequence of elevated cytokinin levels was also found in detached leaves (8-fold after 4 d) and in roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5-8.5), its activity
in vitro
was enhanced fourfold in the presence of copper-imidazole and the apparent Km(iP) values were in the range of 3.1 ╡M to 4.9 ╡M. The increase in cytokinin oxidase activity in cytokinin overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues which may contribute to hormone homeostasis.
