Cytokinins are classical plant hormones that control numerous developmental processes throughout the plant life cycle. Cytokinin-responsive cells activate transcription via a phospho-relay signaling network. Type-B nuclear RESPONSE REGULATOR (RR) proteins mediate transcriptional activation as the final step in the signaling cascade. They bind to promoters of immediate-early target genes via a conserved Myb-related DNA-binding domain. To monitor transcriptional activation in response to a cytokinin stimulus, we have constructed a synthetic promoter, TCS (two-component signaling sensor) that harbors the concatemerized binding motifs for activated type-B RR in an optimized configuration. Here, we describe our protocols for imaging TCSn::GFP expression in transgenic Arabidopsis plants. The use of the fluorescent reporter GFP allows the visualization of cytokinin-responding cells by fluorescent microscopy without the need for tissue processing steps, or staining reactions. This method is fast and with a low risk of artifacts. However, since cytokinin signaling integrates various environmental information including light, nutrient status, and biotic and abiotic stress, special care needs to be devoted to the control of growth conditions.
Keywords: Arabidopsis thaliana; Cytokinin signaling; Fluorescent microscopy; Plant development; Plant physiology; Synthetic reporter.