Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.
- MeSH
- Arabidopsis cytology genetics metabolism MeSH
- Brefeldin A pharmacology MeSH
- Cell Membrane metabolism MeSH
- Cytokinins chemistry metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Fluorescent Dyes chemistry metabolism MeSH
- Plants, Genetically Modified MeSH
- Meristem cytology metabolism MeSH
- Protein Kinases genetics metabolism MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Receptors, Cell Surface genetics metabolism MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Signal Transduction drug effects MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cytokinins and ethylene control plant development via sensors from the histidine kinase (HK) family. However, downstream signaling pathways for the key phytohormones are distinct. Here we report that not only cytokinin but also ethylene is able to control root apical meristem (RAM) size through activation of the multistep phosphorelay (MSP) pathway. We found that both cytokinin and ethylene-dependent RAM shortening requires ethylene binding to ETR1 and the HK activity of ETR1. The receiver domain of ETR1 interacts with MSP signaling intermediates acting downstream of cytokinin receptors, further substantiating the role of ETR1 in MSP signaling. We revealed that both cytokinin and ethylene induce the MSP in similar and distinct cell types with ETR1-mediated ethylene signaling controlling MSP output specifically in the root transition zone. We identified members of the MSP pathway specific and common to both hormones and showed that ETR1-regulated ARR3 controls RAM size. ETR1-mediated MSP spatially differs from canonical CTR1/EIN2/EIN3 ethylene signaling and is independent of EIN2, indicating that both pathways can be spatially and functionally separated. Furthermore, we demonstrated that canonical ethylene signaling controls MSP responsiveness to cytokinin specifically in the root transition zone, presumably via regulation of ARR10, one of the positive regulators of MSP signaling in Arabidopsis.
- MeSH
- Arabidopsis cytology drug effects growth & development metabolism MeSH
- Cytokinins metabolism pharmacology MeSH
- Ethylenes metabolism pharmacology MeSH
- Phosphorylation drug effects MeSH
- Plant Roots drug effects growth & development MeSH
- Arabidopsis Proteins metabolism MeSH
- Receptors, Cell Surface metabolism MeSH
- Signal Transduction drug effects MeSH
- Dose-Response Relationship, Drug MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.
- MeSH
- Arabidopsis enzymology genetics MeSH
- Crystallography, X-Ray MeSH
- Nuclear Magnetic Resonance, Biomolecular MeSH
- Protein Kinases chemistry genetics MeSH
- Protein Domains MeSH
- Arabidopsis Proteins chemistry genetics MeSH
- Receptors, Cell Surface chemistry genetics MeSH
- Protein Structure, Secondary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.
- MeSH
- Arabidopsis genetics metabolism radiation effects MeSH
- Cytokinins metabolism MeSH
- Phytochrome A genetics metabolism MeSH
- Plants, Genetically Modified MeSH
- Heme Oxygenase (Decyclizing) genetics metabolism MeSH
- Hypocotyl genetics metabolism radiation effects MeSH
- Models, Genetic MeSH
- Mutation MeSH
- Protein Kinases genetics metabolism MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Gene Expression Regulation, Plant genetics radiation effects MeSH
- Signal Transduction genetics radiation effects MeSH
- Light * MeSH
- Publication type
- Journal Article MeSH
The multistep phosphorelay (MSP) is a central signaling pathway in plants integrating a wide spectrum of hormonal and environmental inputs and controlling numerous developmental adaptations. For the thorough comprehension of the molecular mechanisms underlying the MSP-mediated signal recognition and transduction, the detailed structural characterization of individual members of the pathway is critical. In this review we describe and discuss the recently known crystal and nuclear magnetic resonance structures of proteins acting in MSP signaling in higher plants, focusing particularly on cytokinin and ethylene signaling in Arabidopsis thaliana. We discuss the range of functional aspects of available structural information including determination of ligand specificity, activation of the receptor via its autophosphorylation, and downstream signal transduction through the phosphorelay. We compare the plant structures with their bacterial counterparts and show that although the overall similarity is high, the differences in structural details are frequent and functionally important. Finally, we discuss emerging knowledge on molecular recognition mechanisms in the MSP, and mention the latest findings regarding structural determinants of signaling specificity in the Arabidopsis MSP that could serve as a general model of this pathway in all higher plants.
Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.
- MeSH
- Cytokinins pharmacology MeSH
- Catalytic Domain MeSH
- Proteasome Endopeptidase Complex metabolism MeSH
- Arabidopsis Proteins chemistry drug effects metabolism MeSH
- Proteolysis drug effects MeSH
- Repressor Proteins chemistry metabolism MeSH
- Protein Stability MeSH
- Transcription Factors metabolism MeSH
- Publication type
- Journal Article MeSH
Integrating important environmental signals with intrinsic developmental programmes is a crucial adaptive requirement for plant growth, survival, and reproduction. Key environmental cues include changes in several light variables, while important intrinsic (and highly interactive) regulators of many developmental processes include the phytohormones cytokinins (CKs) and ethylene. Here, we discuss the latest discoveries regarding the molecular mechanisms mediating CK/ethylene crosstalk at diverse levels of biosynthetic and metabolic pathways and their complex interactions with light. Furthermore, we summarize evidence indicating that multiple hormonal and light signals are integrated in the multistep phosphorelay (MSP) pathway, a backbone signalling pathway in plants. Inter alia, there are strong overlaps in subcellular localizations and functional similarities in components of these pathways, including receptors and various downstream agents. We highlight recent research demonstrating the importance of CK/ethylene/light crosstalk in selected aspects of plant development, particularly seed germination and early seedling development. The findings clearly demonstrate the crucial integration of plant responses to phytohormones and adaptive responses to environmental cues. Finally, we tentatively identify key future challenges to refine our understanding of the molecular mechanisms mediating crosstalk between light and hormonal signals, and their integration during plant life cycles.
Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.
- MeSH
- Arabidopsis metabolism MeSH
- X-Ray Diffraction MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Phosphotransferases chemistry isolation & purification MeSH
- Crystallization MeSH
- Arabidopsis Proteins chemistry isolation & purification MeSH
- Signal Transduction * MeSH
- Transition Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Multistep phosphorelay (MSP) pathways mediate a wide spectrum of adaptive responses in plants, including hormonal and abiotic stress regulations. Recent genetic evidence suggests both partial redundancy and possible functional cross-talk on the one hand and a certain level of specificity on the other. Here, we discuss recent achievements improving our understanding of possible molecular mechanisms of specificity in MSP. We consider a certain evolutionary conservation of ancestral two-component signalling systems from bacteria in a process of molecular recognition that, as we have recently shown, could be applied also to a certain extent in the case of plant MSP. Furthermore, we discuss possible roles of kinase and phosphatase activities, kinetics of both these enzymatic reactions, and phosphorylation lifetime. We include also recent findings on the expression specificity of individual members of MSP pathways and, finally, based on our recent findings, we speculate about a possible role of magnesium in regulation of MSP pathways in plants. All these mechanisms could significantly influence specificity and signalling output of the MSP pathways.
Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.
- MeSH
- Arabidopsis enzymology genetics physiology MeSH
- Phosphorylation MeSH
- Phosphotransferases genetics metabolism MeSH
- Histidine metabolism MeSH
- Crystallography, X-Ray MeSH
- Protein Interaction Mapping MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Protein Kinases chemistry genetics isolation & purification metabolism MeSH
- Arabidopsis Proteins chemistry genetics isolation & purification metabolism MeSH
- Recombinant Fusion Proteins MeSH
- Sensitivity and Specificity MeSH
- Signal Transduction physiology MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
