Biochemical characterization of plant hormone cytokinin-receptor histidine kinases using microorganisms.

Results of recent studies on the model higher plant Arabidopsis thaliana have led us to learn about the generality and versatility of two-component systems (TCS) in eukaryotes. In the plant, TCS are crucially involved in certain signal transduction mechanisms underlying the regulation of plant development in response to a subset of plant hormones, namely, cytokinin and ethylene. Results of extensive plant genomics revealed that these hormone-responsive TCS are evolutionarily conserved in many other plants, including mosses, grasses, crops, and trees. In particular, the conserved cytokinin-responsive TCS is typical in the sense that the signaling pathway consists of cytokinin-receptor histidine kinases (HK), histidine-containing phosphotransfer (HPt) factors, and downstream phosphoaccepting response regulators (RR), which together act as His-to-Asp multistep phosphorelay components, and which together modulate the downstream network of cytokinin-responsive gene regulation. The ethylene-responsive TCS is atypical in that ethylene-receptor HKs appear to directly interact with the downstream mitogen-activated protein kinase (MAPK) cascade. The ethylene-responsive HKs have already been introduced in the previous edition of Methods in Enzymology [Schaller, G. E., and Binder, B. M. (2007). Biochemical characterization of plant ethylene receptors following transgenic expression in yeast. Methods Enzymol. 422, 270-287]. Hence, here we focus on the cytokinin-receptor HKs, which are capable of functioning in microorganisms, such as Escherichia coli and Saccharomyces cerevisiae. Some versatile protocols useful for analyzing plant TCS factors by employing these microorganisms will be introduced.