A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta.

Cytokinins are classic plant hormones that orchestrate plant growth, development, and physiology. They affect gene expression in target cells by activating a multistep phosphorelay network. Type-B response regulators, acting as transcriptional activators, mediate the final step in the signaling cascade. Previously, we have introduced a synthetic reporter, Two Component signaling Sensor (TCS)::green fluorescent protein (GFP), which reflects the transcriptional activity of type-B response regulators. TCS::GFP was instrumental in uncovering roles of cytokinin and deepening our understanding of existing functions. However, TCS-mediated expression of reporters is weak in some developmental contexts where cytokinin signaling has a documented role, such as in the shoot apical meristem or in the vasculature of Arabidopsis (Arabidopsis thaliana). We also observed that GFP expression becomes rapidly silenced in TCS::GFP transgenic plants. Here, we present an improved version of the reporter, TCS new (TCSn), which, compared with TCS, is more sensitive to phosphorelay signaling in Arabidopsis and maize (Zea mays) cellular assays while retaining its specificity. Transgenic Arabidopsis TCSn::GFP plants exhibit strong and dynamic GFP expression patterns consistent with known cytokinin functions. In addition, GFP expression has been stable over generations, allowing for crosses with different genetic backgrounds. Thus, TCSn represents a significant improvement to report the transcriptional output profile of phosphorelay signaling networks in Arabidopsis, maize, and likely other plants that display common response regulator DNA-binding specificities.