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

Abstract

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast 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 detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).