Chemically induced expression of the rolC-encoded beta-glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta

The rolC gene of Agrobacterium rhizogenes T-DNA plays an essential role 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-glucosidase is due to an alteration of the cytokinin balance in planta, HPLC radiocounting assays of [H-3]-labeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia 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 NS-glucosides was not detected at substrate concentrations close 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 the gene was expressed under the transcriptional control of a modified tetracycline-inducible 35S promoter, This was done to avoid 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 also accumulated high levels of cytokinin O-glucosides. It is concluded 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.