Multistep phosphorelay (MSP) signaling integrates hormonal and environmental signals to control both plant development and adaptive responses. Type-A RESPONSE REGULATOR (RRA) genes, the downstream members of the MSP cascade and cytokinin primary response genes, are thought to mediate primarily the negative feedback regulation of (cytokinin-induced) MSP signaling. However, transcriptional data also suggest the involvement of RRA genes in stress-related responses. By employing evolutionary conservation with the well-characterized Arabidopsis thaliana RRA genes, we identified five and 38 novel putative RRA genes in Brassica oleracea and Brassica napus, respectively. Our phylogenetic analysis suggests the existence of gene-specific selective pressure, maintaining the homologs of ARR3, ARR6, and ARR16 as singletons during the evolution of Brassicaceae. We categorized RRA genes based on the kinetics of their cytokinin-mediated up-regulation and observed both similarities and specificities in this type of response across Brassicaceae species. Using bioinformatic analysis and experimental data demonstrating the cytokinin and abiotic stress responsiveness of the A. thaliana-derived TCSv2 reporter, we unveil the mechanistic conservation of cytokinin- and stress-mediated up-regulation of RRA genes in B. rapa and B. napus. Notably, we identify partial cytokinin dependency of cold stress-induced RRA transcription, thus further demonstrating the role of cytokinin signaling in crop adaptive responses.

• Keywords
• Arabidopsis thaliana, Brassica napus, Brassica oleracea, Brassica rapa, cytokinins, multistep phosphorelay, osmotic stress, salinity, two-component signaling, type-A response regulator,
• MeSH
• Arabidopsis genetics   physiology   metabolism   MeSH
• Brassica napus genetics   physiology   metabolism   MeSH
• Brassica * genetics   physiology   metabolism   MeSH
• Cytokinins * metabolism   MeSH
• Phylogeny MeSH
• Stress, Physiological * MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Proteins * genetics   metabolism   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokinins * MeSH
• Plant Growth Regulators MeSH
• Plant Proteins * MeSH

Plants growing in any particular geographical location are exposed to variable and diverse environmental conditions throughout their lifespan. The multifactorial environmental pressure resulted into evolution of plant adaptation and survival strategies requiring ability to integrate multiple signals that combine to yield specific responses. These adaptive responses enable plants to maintain their growth and development while acquiring tolerance to a variety of environmental conditions. An essential signaling cascade that incorporates a wide range of exogenous as well as endogenous stimuli is multistep phosphorelay (MSP). MSP mediates the signaling of essential plant hormones that balance growth, development, and environmental adaptation. Nevertheless, the mechanisms by which specific signals are recognized by a commonly-occurring pathway are not yet clearly understood. Here we summarize our knowledge on the latest model of multistep phosphorelay signaling in plants and the molecular mechanisms underlying the integration of multiple inputs including both hormonal (cytokinins, ethylene and abscisic acid) and environmental (light and temperature) signals into a common pathway. We provide an overview of abiotic stress responses mediated via MSP signaling that are both hormone-dependent and independent. We highlight the mutual interactions of key players such as sensor kinases of various substrate specificities including their downstream targets. These constitute a tightly interconnected signaling network, enabling timely adaptation by the plant to an ever-changing environment. Finally, we propose possible future directions in stress-oriented research on MSP signaling and highlight its potential importance for targeted crop breeding.

• Keywords
• Arabidopsis, abiotic stress, abscisic acid, cytokinin, ethylene, light signaling, multistep phosphorelay (MSP), temperature,
• Publication type
• Journal Article MeSH
• Review 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.

• Keywords
• X-ray crystallography, histidine kinase, nuclear magnetic resonance (NMR), protein dynamic, protein phosphorylation, receiver domain, relaxation dispersion,
• 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
• Names of Substances
• CKI1 protein, Arabidopsis MeSH Browser
• ETR1 protein, Arabidopsis MeSH Browser
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface 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
• Names of Substances
• CKI1 protein, Arabidopsis MeSH Browser
• Cytokinins MeSH
• Phytochrome A MeSH
• Heme Oxygenase (Decyclizing) MeSH
• HY1 protein, Arabidopsis MeSH Browser
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

• MeSH
• Arabidopsis MeSH
• Chromatin Immunoprecipitation MeSH
• Cytokinins metabolism   MeSH
• Plants, Genetically Modified MeSH
• Microscopy, Confocal MeSH
• Plant Roots metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Response Elements MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   metabolism   MeSH
• Green Fluorescent Proteins MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CRF3 protein, Arabidopsis MeSH Browser
• CRF6 protein, Arabidopsis MeSH Browser
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• PIN1 protein, Arabidopsis MeSH Browser
• PIN7 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Transcription Factors MeSH
• Green Fluorescent Proteins MeSH

Dye perfusion is commonly used for the identification of conductive elements important for the study of xylem development as well as precise hydraulic estimations. The tiny size of inflorescence stems, the small amount of vessels in close arrangement, and high hydraulic resistivity delimit the use of the method for quantification of the water conductivity of Arabidopsis thaliana, one of the recently most extensively used plant models. Here, we present an extensive adjustment to the method in order to reliably identify individual functional (conductive) vessels. Segments of inflorescence stems were sealed in silicone tubes to prevent damage and perfused with a dye solution. Our results showed that dyes often used for staining functional xylem elements (safranin, fuchsine, toluidine blue) failed with Arabidopsis. In contrast, Fluorescent Brightener 28 dye solution perfused through segments stained secondary cell walls of functional vessels, which were clearly distinguishable in native cross sections. When compared to identification based on the degree of development of secondary cell walls, identification with the help of dye perfusion revealed a significantly lower number of functional vessels and values of theoretical hydraulic conductivity. We found that lignified but not yet functional vessels form a substantial portion of the xylem in apical and basal segments of Arabidopsis and, thus, significantly affect the analyzed functional parameters of xylem. The presented methodology enables reliable identification of individual functional vessels, allowing thus estimations of hydraulic conductivities to be improved, size distributions and vessel diameters to be refined, and data variability generally to be reduced.

• Keywords
• Fluorescent Brightener 28, conductive elements, dye perfusion, fluorescence, hydraulic conductivity, vessel, xylem,
• Publication type
• Journal Article MeSH

Plant vascular meristems are sets of pluripotent cells that enable radial growth by giving rise to vascular tissues and are therefore crucial to plant development. However, the overall dynamics of cellular determination and patterning in and around vascular meristems is still unexplored. We study this process in the shoot vascular tissue of Arabidopsis thaliana, which is organized in vascular bundles that contain three basic cell types (procambium, xylem and phloem). A set of molecules involved in this process has now been identified and partially characterized, but it is not yet clear how the regulatory interactions among them, in conjunction with cellular communication processes, give rise to the steady patterns that accompany cell-fate determination and arrangement within vascular bundles. We put forward a dynamic model factoring in the interactions between molecules (genes, peptides, mRNA and hormones) that have been reported to be central in this process, as well as the relevant communication mechanisms. When a few proposed interactions (unverified, but based on related data) are postulated, the model reproduces the hormonal and molecular patterns expected for the three regions within vascular bundles. In order to test the model, we simulated mutant and hormone-depleted systems and compared the results with experimentally reported phenotypes. The proposed model provides a formal framework integrating a set of growing experimental data and renders a dynamic account of how the collective action of hormones, genes, and other molecules may result in the specification of the three main cell types within shoot vascular bundles. It also offers a tool to test the necessity and sufficiency of particular interactions and conditions for vascular patterning and yields novel predictions that may be experimentally tested. Finally, this model provides a reference for further studies comparing the overall dynamics of tissue organization and formation by meristems in other plant organs and species.

• MeSH
• Algorithms MeSH
• Arabidopsis cytology   genetics   metabolism   MeSH
• Models, Biological MeSH
• Cell Differentiation MeSH
• Plant Vascular Bundle cytology   genetics   metabolism   MeSH
• Cytokinins physiology   MeSH
• Gene Regulatory Networks MeSH
• Meristem cytology   genetics   metabolism   MeSH
• Computer Simulation MeSH
• Protein Kinases physiology   MeSH
• Arabidopsis Proteins physiology   MeSH
• Gene Expression Regulation, Plant MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH
• PXY protein, Arabidopsis MeSH Browser

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.

• Keywords
• AHP2, Arabidopsis thaliana, histidine phosphotransfer proteins, multi-step phosphorelay,
• 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
• Names of Substances
• AHP2 protein, Arabidopsis MeSH Browser
• Phosphotransferases MeSH
• Arabidopsis Proteins MeSH