Chemically induced expression of the rolC encoded ▀-glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta
Martin Faiss
1
, Miroslav Strnad
2
, Pascale Redig
3
, Karel Dolezal
2
, Jan Hanus4, Harry Van Onckelen
3
, Thomas Schmülling
1
Plant J 10: 33-46.
1
UniversitΣt Tübingen, Lehrstuhl fⁿr Allgemeine Genetik, Auf der Morgenstelle 28, D-72076 Tübingen, Germany,
2
Institute of Experimental Botany, Department of Plant Biotechnology, Sokolovská 6, CZ-77200 Olomouc, Czech Republic,
3
University of Antwerpen, Department of Biology, Universiteitsplein 1, B-2610 Antwerp, Belgium, and
4
Institute of Experimental Botany, Isotope Laboratory, Videnská 1083, CZ-14220 Prague, Czech Republic
Abstract
The rolC gene of
Agrobacterium rhizogenes
T-DNA plays an essential role in the establishment of the hairy root disease and its overexpression causes in transgenic plants pleiotropic developmental alterations. We have investigated whether the biological activity of the
rolC
ß-glucosidase is due to an alteration of the cytokinin balance
in planta
. HPLC radiocounting assays of [
3
H]-labelled cytokinin glucosides fed exogenously to tobacco leaf disks, to
rolC
expressing
E. coli
cells or cell free extracts showed that cytokinin N3- and O-glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7- and N9-glucosides was not detected at substrate concentrations closed to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to
rolC
expressing tissues. For analysis of the
rolC
activity on endogenous cytokinin-conjugates we have expressed the gene under the transcriptional control of a modified tetracycline-inducible 35S promoter. This was done to avoid the possible interference with secondary effects or plant homeostatic mechanisms which could mask primary
in planta
events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the
rolC
gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin-synthezising
ipt
gene and the
rolC
gene showed added phenotypes indicating that the
rolC
phenotype is mediated on a signalling pathway different from those of cytokinins.
RolC/ipt
hybrids accumulated also high levels of cytokinin O-glucosides. We conclude that the phenotypic alterations caused by the rolC gene product are not due to a release of free cytokinins from inactive conjugates, most likely because of subcellular compartmentation of the putative substrate.
