CHEMICALLY-INDUCED EXPRESSION OF THE ROLC-ENCODED BETA-GLUCOSIDASE INTRANSGENIC TOBACCO PLANTS AND ANALYSIS OF CYTOKININ METABOLISM - ROLCDOES NOT HYDROLYZE ENDOGENOUS CYTOKININ GLUCOSIDES IN PLANTA

The rolC gene of Agrobacterium rhizogenes T-DNA plays an essential rol e in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations, This study investigated whether the biological activity of the rolC beta-gl ucosidase is due to an alteration of the cytokinin balance in planta, HPLC radiocounting assays of [H-3]-labeled cytokinin glucosides fed ex ogenously to tobacco leaf disks, to rolC expressing Escherichia coil c ells or cell-free extracts showed that cytokinin N3- and O-glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7- and NS-glucosides was not detected at substrate concentrations close to p hysiological levels. Furthermore, these conjugates were also not activ e as cytokinins in biotests when fed to rolC-expressing tissues. For a nalysis of the rolC activity on endogenous cytokinin conjugates the ge ne was expressed under the transcriptional control of a modified tetra cycline-inducible 35S promoter, This was done to avoid possible interf erence with secondary effects or plant homeostatic mechanisms which co uld mask primary in planta events when transgenes are expressed consti tutively. No changes in the endogenous pool of different cytokinin glu cosides, as determined by a newly developed electrospray tandem mass s pectroscopy directly coupled to high performance liquid chromatography , were found following chemical induction of the rolC gene. Also the l evels of free cytokinins remained unchanged after gene induction, Hybr id tobacco plants expressing the cytokinin-synthezising ipt gene and t he rolC gene showed added phenotypes indicating that the rolC phenotyp e is mediated on a signalling pathway different from those of cytokini ns, RolC/ipt hybrids also accumulated high levels of cytokinin O-gluco sides. It is concluded that the phenotypic alterations caused by the r olC gene product are not due to a release of free cytokinins from inac tive conjugates, most likely because of subcellular compartmentation o f the putative substrate.