Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role of exogenously supplied auxin and cytokinin on the level and activity of Cdc2-related pro tein kinases and progression through the first cell division cycle after re -activation of cell division. Among the three alfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE) showed only significant activity du ring the first four days of protoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases (PPTTLRE) exhibited only low or undetectable activity, respectively, during this period. Although the Cdc2MsA/B protein could be d etected in leaves and freshly isolated protoplasts in variable amounts, the kinase was never active in these cells. The kinase protein disappeared fro m protoplast-derived cells at the beginning (8h) of culture but its synthes is re-commenced dependent on the presence of exogenous auxin but not cytoki nin. The cytokinin response of alfalfa protoplast-derived cells varied sign ificantly in different experiments although cytokinin was always required f or completion of the first cell division cycle. Frequently both auxin and c ytokinin was required for DNA replication as not more than 5% of cells coul d incorporate BrdU into their DNA during three days and significant Cdc2MsA /B activity could not be detected in the absence of exogenous cytokinin. In other protoplast populations, the Cdc2MsA/B kinase was activated by auxin alone and allowed the protoplast-derived cells to enther the S-phase at a s imilar rate observed in parallel cultures with both auxin and cytokinin. Ev en in these cultures, however, ca. 95% of the protoplast-derived cells were arrested before mitosis without exogenous cytokinin supply which could be correlated with decreasing Cdc2MsA/B activity. These observations suggest, that although cytokinin is required for both G0-G1/S and G2/M cell cycle tr ansitions, in certain cultures the G1/S requirement is overcome by some unk nown factors (e.g. conditions of explants; endogenous cytokinins etc.). Fur thermore, our experiments indicate, that the roles of cytokinin are related to the post-translational regulation of the Cdc2MsA/B kinase complex at bo th cell cycle transition points in alfalfa leaf protoplast-derived cells. F inally, as a marker for the transition from the differentiated (G0) stage t o the activated (G1) stage, we suggest using the parameters of nuclear morp hology (size and ratio of nucleus/nucleolus).