Hormonal control of gene expression during reactivation of the cell cycle in tobacco mesophyll protoplasts

The roles of auxin and cytokinin in cell cycle reactivation were studied du ring the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacu m. Using hormone delay and withdrawal studies we found that auxin was requi red by 0-4 h of culture, whereas cytokinin was not required until hour 10-1 2, which is 6-10 h before S phase. Cycloheximide blocks division, indicatin g that protein synthesis is required. In an effort to detect a molecular re sponse to either hormone, we examined the expression of the cell cycle mark er, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. H owever, cdc2 was partially induced by either auxin or cytokinin alone, sugg esting that cdc2 expression is not the primary target of either hormone. Ou r hormone delay experiments suggest that there are separate signal transduc tion pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechan isms for these distinct pathways remain to be elucidated.