While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.

Biozentrum Klein Flottbek Universität Hamburg Ohnhorststr 18 D 22609 Hamburg Germany

Institut Jean Pierre Bourgin UMR1318 INRA AgroParisTech INRA Centre de Versailles Grignon Route de St Cyr 78026 Versailles Cedex France

Institute for Biology Applied Genetics Dahlem Centre of Plant Sciences Freie Universität Berlin Albrecht Thaer Weg 6 D 14195 Berlin Germany

Institute for Biology Applied Genetics Dahlem Centre of Plant Sciences Freie Universität Berlin Albrecht Thaer Weg 6 D 14195 Berlin Germany Biology Department Adelphi University Science 116 1 South Avenue PO Box 701 Garden City NY 11530 070 USA

Laboratory of Growth Regulators and Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Palacký University and Institute of Experimental Botany ASCR Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

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