A Robust and Sensitive Synthetic Sensor to Monitor the Transcriptional Output of the Cytokinin Signaling Network in Planta      

Abstract

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.

Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL

“…By contrast, the signals of the cytokinin response remain undetectable in the prospective SAM until the heart stage of the embryo, which is much later than the initiation of WUS expression (Müller and Sheen, 2008;Cheng et al, 2013;Zürcher et al, 2013). It is likely that cytokinin signaling acts upstream of WUS during shoot regeneration and downstream of WUS in embryonic SAM establishment.…”

Synthetic Botany

“…Examples include a light-inducible gene expression system activated by red light and switched off by far-red light (Müller et al 2014) as well as a range of in vivo biosensors to monitor levels of cytokinin (Müller and Sheen 2008;Zurcher et al 2013), auxin (Wend et al 2013), plant pathogens (Liu et al 2011;Fethe et al 2014), and elicitors (Koschmann et al 2012). These are examples of synthetic circuits that have been constructed in plants using methods for DNA manipulation, transgene regulation, transformation, and analysis to yield basic systems for chassis engineering and genetic network design in plant synthetic biology.…”

“…Traits of agricultural importance successfully introduced to plants using recombinant DNA technology include herbicide resistance (Comai et al 1985), drought resistance (Kumar et al 2014), pest resistance (Bates et al 2005), pathogen resistance (Brunner et al 2011;Horvath et al 2012;Jones et al 2014), abiotic stress resistance (Jaglo-Ottosen et al 1998), enhanced photosynthetic capacity (Ku et al 2001), im-proved nitrogen use efficiency (Yanagisawa et al 2004), and added nutritional value (Ye et al 2000). However, although plants hold a unique promise for bioproduction at the gigatonne scale, efforts in genetic engineering of plants are lagging compared with microbial systems (Antunes et al 2009(Antunes et al , 2011Liu et al 2011Liu et al , 2013Koschmann et al 2012;Wend et al 2013;Zurcher et al 2013;Fethe et al 2014;Mül-ler et al 2014).…”

Externally imposed electric field enhances plant root tip regeneration

“…2013) and generated temporal series of individual regenerating roots in the same conditions used for the auxin reporter R2D2 . In uncut roots, the signal was strongest in columella cells, lateral root cap and vasculature initials, with no obvious difference between roots exposed to the field or in mock conditions (Fig.…”

“…2004), and TCSn::GFP (Zürcher et al. 2013) are in wild‐type Arabidopsis thaliana Columbia (Col‐0) ecotype background. The reporter line expressing RPS5A::mDII:ntdTomato‐RPS5A::DII:n3Venus , also known as R2D2 reporter (Liao et al.…”