Expression of Spcdc25 in Arabidopsis alters the response of hypocotyl explants to auxin & cytokinin in culture

The eukaryotic cell cycle comprises four distinct phases, mitosis (M), G1, DNA synthesis (S)-phase and G2 in which the key transitions are G1/S and G2/M. Progression of dividing cells through the cell cycle is controlled by key enzymes: cyclin-dependent kinases (CDKs), the products of cdc2-like genes. The regulation of CDK activity includes, amongst others, its activation by Cdc25 phosphatase at G2/M. This positive regulator is balanced by negative regulation by a protein kinase encoded by wee1. A plant homologue of the inactivating kinase, wee1, has already been cloned. However, a homologue of the activating phosphatase cdc25 has not been unequivocally identified in plants yet. The expression of cdc2-like genes can be influenced by many factors including plant growth regulators, mainly auxins and cytokinins. Expressing the fission yeast cdc25 (Spcdc25) in the tobacco BY2 cell line results in cells bypassing a cytokinin block, and in explants of tobacco expressing Spcdc25, shoot development is stimulated not only in a treatment that favours shoot formation (high cytokinin and low auxin) but also under root-stimulating conditions (high auxin and low cytokinin) and even without exogenous growth regulator treatment. Wild type Arabidopsis hypocotyls explants respond to increasing levels of cytokinin with rapid proliferation, greening and formation of shoots, but are unable to form shoots in the absence of auxin and cytokinin. We tested if hypocotyl explants from Arabidopsis transformed with constitutive and inducible lines Spcdc25 respond like wt to auxin (NAA) and cytokinin (Kinetin) in a system of grids in which we increase concentration of these exogenous plant growth regulators. We find that Wt and Spcdc25 hypocotiles segments are very sensible to higher liveles of ormons (1000 ngml-1 to 3000 ngml-1), calli grew with a lot of root hairs and the analysis of shots formation was impossible. Also is required a minimum level of NAA for calli induction in these strains: at 25 ngml-1 NAA/Kinetin we start to see grow of calli in Spcdc25 lines, plants in WT, not grow in not induced Spcdc25 line. Interesting results at 25, 50 ngml-1 NAA and 200ngml-1 Kinetin where in Spcdc25 there is grow of real roots, not visible in wt and not induced Spcdc25 line. At several concentrations of hormones Spcdc25 forms buds they will be show and discussed