J. Plant Physiol. 158. 1117–1123 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp The requirements for Ca 2 + , protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L. Sonali P. Barwe, Muthukrishnan Sathiyabama, Chelliah Jayabaskaran* Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received January 12, 2001 · Accepted May 21, 2001 Summary We have previously reported that both Ca 2 + and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induc- tion of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark- grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exoge- nous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumula- tion of CACHT mRNA was inhibited by a plasma membrane Ca 2 + channel blocker verapamil. Treat- ment of cotyledons with exogenous CaCl 2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of stauro- sporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involve- ment of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca 2 + influx from extracellular space, protein phosphoryla- tion, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation. Key words: Ca 2 + – chitinase-cytokinin signaling – Cucumis sativus L. – protein phosphorylation – transcript level – zeatin Abbreviations: BA benzyladenine. – CACHT cucumber acidic chitinase. – CHX cycloheximide. – EGTA ethylene glycol. – bis(2-amino-ethylether) tetraacetic acid. – K kinetin. – NaF sodium fluoride. – ST staurosporine. – Z zeatin. – ZR zeatin riboside * E-mail corresponding author: cjb@biochem.iisc.ernet.in 0176-1617/01/158/09 –1117 $ 15.00/0