Plant stem-cell pools, the source for all organs, are first established during embryogenesis. It has been known for decades that cytokinin and auxin interact to control organ regeneration in cultured tissue 1 . Auxin plays a critical role for root-stem cell specification in zygotic embryogenesis 2,3 , but the early embryonic function of cytokinin is obscure 4-6 . Here, we introduce a synthetic reporter to universally visualise cytokinin output in vivo. Surprisingly, the first embryonic signal is detected in the hypophysis, the founder cell of the root stem-cell system. Its apical daughter cell, precursor of the quiescent centre, maintains phosphorelay activity, whereas the basal daughter cell represses signalling output. Intriguingly, auxin activity levels exhibit the inverse profile. We show that auxin antagonizes cytokinin output in the basal cell-lineage by direct transcriptional activation of ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, ARR7 and ARR15, feedback repressors of cytokinin signalling. Loss of ARR7 and ARR15 function or ectopic cytokinin signalling in the basal cell during early embryogenesis results in a defective root-stem cell system. The results provide a molecular model of transient and antagonistic interaction between auxin and cytokinin critical for specifying the first root-stem cell niche.Cytokinins are adenine-derived signalling molecules that play many essential roles in postembryonic growth and development. However, the role of cytokinin signalling in early embryogenesis remained unclear 4-6 . To visualise cytokinin's signalling output in vivo, we aimed at designing a synthetic reporter that overcomes the limitations of current reporters, typically immediate-early cytokinin target genes. The discrete expression patterns of these markers 7,8 indicate that they integrate unknown secondary input that reflects cytokininindependent regulation. Cytokinin signalling is mediated by a multistep two-component circuitry through histidine and aspartate phosphorelay 9 . Nuclear B-type response regulators (RRs) mediate transcriptional activation in response to phosphorelay signalling activity, while A-type RRs repress signalling in a negative-feedback loop. The DNA-binding domains of diverse B-type RR family members are conserved and bind a common DNA-target sequence (A/G)GAT(T/C) in vitro 10-12 . This motif is significantly enriched in the cis-regulatory region of immediate-early cytokinin target genes 13 , suggesting its in vivo relevance. To generate a universal cytokinin reporter we tested and optimised synthetic reporter designs using luciferase (LUC) activity in Arabidopsis mesophyll protoplast assays 14,15 . The resulting synthetic reporter TCS-LUC (TwoComponent-output-Sensor) harboured the concatemerised B-type ARR-binding motifs 10-12 and a minimal 35S promoter 14 . Only cytokinins activated TCS-LUC, while other plantCorrespondence and requests for materials should be addressed to J.S. (e-mail: sheen@molbio.mgh.harvard.edu) and B.M. (e-mail: mueller@molbio.mgh.harvard.edu). The autho...

Despite long-standing observations on diverse cytokinin actions, the discovery path to cytokinin signaling mechanisms was tortuous. Unyielding to conventional genetic screens, experimental innovations were paramount in unraveling the core cytokinin signaling circuitry, which employs a large repertoire of genes with overlapping and specific functions. The canonical two-component transcription circuitry involves His kinases that perceive cytokinin and initiate signaling, as well as His-to-Asp phosphorelay proteins that transfer phosphoryl groups to response regulators, transcriptional activators, or repressors. Recent advances have revealed the complex physiological functions of cytokinins, including interactions with auxin and other signal transduction pathways. This review begins by outlining the historical path to cytokinin discovery and then elucidates the diverse cytokinin functions and key signaling components. Highlights focus on the integration of cytokinin signaling components into regulatory networks in specific contexts, ranging from molecular, cellular, and developmental regulations in the embryo, root apical meristem, shoot apical meristem, stem and root vasculature, and nodule organogenesis to organismal responses underlying immunity, stress tolerance, and senescence.

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.

The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. We have uncovered a simple mechanism through which Dpp, a TGFbeta/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. Our findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation.

Morphogen gradients control body pattern by differentially regulating cellular behavior. Here, we analyze the molecular events underlying the primary response to the Dpp/BMP morphogen in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker (brk) gene. We first provide significance for the brk expression gradient by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. We then dissect the brk regulatory region and identify two separable elements with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, we present genetic and biochemical evidence that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, our results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression.

Morphogenetic signals control patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. Here, we show that the responses to cytokinin signaling during Arabidopsis development are constrained by the transporter PURINE PERMEASE 14 (PUP14). PUP14 is inversely expressed to the cytokinin signaling readout. The loss of PUP14 function allows ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots and the shoot apical meristem. PUP14 protein localizes to the plasma membrane and imports bioactive cytokinins, thus depletes apoplastic cytokinin pools and inhibits perception by plasma-membrane localized cytokinin sensors. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape shaping morphogenesis of land plants.

The architecture of a plant's root system, established postembryonically, results from both coordinated root growth and lateral root branching. The plant hormones auxin and cytokinin are central endogenous signaling molecules that regulate lateral root organogenesis positively and negatively, respectively. Tight control and mutual balance of their antagonistic activities are particularly important during the early phases of lateral root organogenesis to ensure continuous lateral root initiation (LRI) and proper development of lateral root primordia (LRP). Here, we show that the early phases of lateral root organogenesis, including priming and initiation, take place in root zones with a repressed cytokinin response. Accordingly, ectopic overproduction of cytokinin in the root basal meristem most efficiently inhibits LRI. Enhanced cytokinin responses in pericycle cells between existing LRP might restrict LRI near existing LRP and, when compromised, ectopic LRI occurs. Furthermore, our results demonstrate that young LRP are more sensitive to perturbations in the cytokinin activity than are developmentally more advanced primordia. We hypothesize that the effect of cytokinin on the development of primordia possibly depends on the robustness and stability of the auxin gradient.

Cytokinins are essential plant hormones that control various processes in plants' development and response to external stimuli. The Arabidopsis cytokinin signal transduction pathway involves hybrid histidine protein kinase sensors, phosphotransfer proteins, and regulators as transcription activators and repressors in a phosphorelay system. Each step is executed by components encoded by multigene families. Recent findings have revealed new functions, new feedback loops, and connections to other signaling pathways.