Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin-and dihydrozeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N-and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis. These f'mdings suggest that zeatin-and dihydrozeatintype cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.

The endogenous levels of the major, naturally occurring cytokinins in fisum safivum ribulose-I ,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin Kglucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3Hlisopentenyladenine and [3Hlisopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. lncorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.

A methodology was developed for the analysis of cytokinins from plant tissue using electrospray tandem mass spectrometry in combination with HPLC. A detection limit of 1 pmol zeatin-riboside injected on-column was achieved under multiple reaction monitoring conditions. Linearity was observed within a concentration range of 1-100 pmol injected on-column. In view of the analysis of a considerable amount of samples, the chromatographic conditions were selected which were a compromise between speed of analysis and resolution. This approach, in combination with multiple reaction monitoring, allowed the analysis of up to 150 samples a day, and quantitation of 16 different cytokinins per sample.

SummaryThe 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 13-glucosidase is due to an alteration of the cytokinin balance in planta.HPLC radiocounting assays of [3H]-Iabeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coil 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 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-

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologous of the homeotic genes DEFA, GLO and PLENA of Antirrhinum majus. Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.