Transient accumulation of cis- and trans-zeatin type cytokinins and its relation to cytokinin oxidase activity during cell cycle of synchronized tobacco BY-2 cells

Changes in levels of endogenous cytokinins and activity of cytokinin oxidase (EC 1.4.3.6) were followed during the cell cycle progression in large-scale (ca. 30 g FW) synchronized culture of the tobacco cell line BY-2. Synchronization was induced either by 4 mM hydroxyurea or 15 μM aphidicolin alone (a single block of the cell cycle at G1/S) or in combination with 1 μM oryzalin (double blocking at G1/S and G2/M). Up to 46% synchrony was obtained. Two distinct cytokinin peaks were detected at 0-2 h and 8.5-9 h after the release from the first block coinciding with the beginning of the S phase and with the onset of mitosis, respectively. Interestingly, as identified by MS/MS, very sharp and transient premitotic peak of cytokinins contained, in addition to trans-zeatin, also considerable amounts of cis-zeatin and zeatin-O-glucoside. Cytokinin oxidase activity exhibited conspicuous maximum parallel to the first peak of cytokinin accumulation and deep minimum corresponding to the culmination of mitotic index. Possible roles of zeatin cis-trans isomerisation, glucosylation and degradation by cytokinin oxidase in regulation of cytokinin levels during the cell cycle progression are discussed.