Blanka Pekárová

The Histidine Kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 Regulate Vascular Tissue Development in Arabidopsis Shoots

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.

Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana.

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.

Structural Aspects of Multistep Phosphorelay-Mediated Signaling in Plants

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.

Cloning, purification, crystallization and preliminary X-ray analysis of the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana

The receiver domain (RD) of a sensor histidine kinase (HK) catalyses the transphosphorylation reaction during the action of HKs in hormonal and abiotic signalling in plants. Crystals of the recombinant RD of the Arabidopsis thaliana HK CYTOKININ-INDEPENDENT1 (CKI1(RD)) have been obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and glycerol as a cryoprotectant. The crystals diffracted to approximately 2.4 A resolution on beamline BW7B of the DORIS-III storage ring. The diffraction improved significantly after the use of a non-aqueous cryoprotectant. Crystals soaked in Paratone-N diffracted to at least 2.0 A resolution on beamline BW7B and their mosaicity decreased more than tenfold. The crystals belonged to space group C222(1), with unit-cell parameters a = 54.46, b = 99.82, c = 79.94 A. Assuming the presence of one molecule of the protein in the asymmetric unit gives a Matthews coefficient V(M) of 2.33 A(3) Da(-1). A molecular-replacement solution has been obtained and structure refinement is in progress.

Molecular Mechanisms of Signalling Specificity Via Phosphorelay Pathways in Arabidopsis

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.

Conformational dynamics as a key factor of signaling mediated by the receiver domain of sensor histidine kinase from Arabidopsis thaliana.

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.

The influence of Mg2+ coordination on 13C and 15N chemical shifts in CKI1RD protein domain from experiment and molecular dynamics/density functional theory calculations

Sequence dependence of 13C and 15N chemical shifts in the receiver domain of CKI1 protein from Arabidopsis thaliana, CKI1RD, and its complexed form, CKI1RD•Mg2+, was studied by means of MD/DFT calculations. MD simulations of a 20–ns production run length were performed. Nine explicitly hydrated structures of increasing complexity were explored, up to a 40‐amino‐acid structure. The size of the model necessary depended on the type of nucleus, the type of amino acid and its sequence neighbors, other spatially close amino acids, and the orientation of amino acid NH groups and their surface/interior position. Using models covering a 10 and a 15 Å environment of Mg2+, a semi‐quantitative agreement has been obtained between experiment and theory for the V67−I73 sequence. The influence of Mg2+ binding was described better by the 15 Å as compared to the 10 Å model. Thirteen chemical shifts were analyzed in terms of the effect of Mg2+ insertion and geometry preparation. The effect of geometry was significant and opposite in sign to the effect of Mg2+ binding. The strongest individual effects were found for 15N of D70, S74, and V68, where the electrostatics dominated; for 13Cβ of D69 and 15N of K76, where the influences were equal, and for 13Cα of F72 and 13Cβ of K76, where the geometry adjustment dominated. A partial correlation between dominant geometry influence and torsion angle shifts upon the coordination has been observed. Proteins 2016; 84:686–699.

Cytokinin and Ethylene Signaling

Cytokinins and ethylene belong to the group of “classical” plant growth regulators controlling a broad spectrum of developmental responses. Models for cytokinin and ethylene signal transduction have been established mainly in Arabidopsis, but the signaling pathways of both phytohormones are believed to be conserved throughout the plant kingdom. Nonetheless, in spite of several decades of intense research, our knowledge on basic principles driving signal recognition and transduction of both phytohormones is still delimited. Cytokinins and ethylene are recognized by proteins from the same family of sensor histidine kinases. However, the mechanism of signal transduction through the (plasma) membrane as well as the downstream members of both signaling cascades differ for cytokinins and ethylene. While cytokinins activate multistep phosphorelay signaling of bacterial origin, ethylene signal is perceived by a series of negative regulations mediated by redundant ethylene sensors and downstream Raf-like kinase.

Antibodies against CKI1RD, a receiver domain of the sensor histidine kinase in Arabidopsis thaliana: from antigen preparation to in planta immunolocalization.

Immunodetection is a powerful tool in functional studies of all organisms. In plants, the gene redundancy and presence of gene families composed of highly homologous members often impedes the unambiguous identification of individual gene products. A family of eight sensor histidine kinases (HKs) mediates the transduction of diverse signals into Arabidopsis thaliana cells, thereby ensuring the initiation of appropriate adaptive responses. Antibodies recognizing specific members of the HK family would be valuable for studying their functions in Arabidopsis and other plant species including important crops. We have focused on developing and applying antibodies against CYTOKININ-INDEPENDENT 1 (CKI1), which encodes a constitutively active membrane-bound sensor HK that regulates the development of female gametophytes and vascular tissue in Arabidopsis. A coding sequence delimiting the C-terminal receiver domain of CKI1 (CKI1(RD)) was expressed in Escherichia coli using the IPTG-inducible expression system and purified to give a highly pure target protein. The purified CKI1(RD) protein was then used as an antigen for anti-CKI1(RD) antibody production. The resulting polyclonal antibodies had a detection limit of 10 ng of target protein at 1:20,000 dilution and were able to specifically distinguish CKI1, both in vitro and in situ, even in a direct comparison with highly homologous members of the same HK family AHK4, CKI2 and ETR1. Finally, anti-CKI1(RD) antibodies were able to selectively bind CKI1-GFP fusion protein in a pull-down assay using crude lysate from an Arabidopsis cell suspension culture. Our results suggest that the receiver domain is a useful target for the functional characterization of sensor HKs in immunological and biochemical studies.

Structure of CKI1RD, the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana

We reported here X-ray structure of the receiver domain of the histidine kinase CKI1 from Arabidopsis thaliana