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.

• Klíčová slova
• crosstalk, cytokinin, ethylene, root development, signaling,
• MeSH
• Arabidopsis cytologie   účinky léků   růst a vývoj   metabolismus   MeSH
• cytokininy metabolismus   farmakologie   MeSH
• ethyleny metabolismus   farmakologie   MeSH
• fosforylace účinky léků   MeSH
• kořeny rostlin účinky léků   růst a vývoj   MeSH
• proteiny huseníčku metabolismus   MeSH
• receptory buněčného povrchu metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cytokininy MeSH
• ethylene MeSH Prohlížeč
• ethyleny MeSH
• ETR1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• receptory buněčného povrchu 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.

• Klíčová slova
• X-ray crystallography, histidine kinase, nuclear magnetic resonance (NMR), protein dynamic, protein phosphorylation, receiver domain, relaxation dispersion,
• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• krystalografie rentgenová MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteinkinasy chemie   genetika   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   genetika   MeSH
• receptory buněčného povrchu chemie   genetika   MeSH
• sekundární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CKI1 protein, Arabidopsis MeSH Prohlížeč
• ETR1 protein, Arabidopsis MeSH Prohlížeč
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH

N6-adenosine methylation (m6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6 A writer proteins in Arabidopsis thaliana. The components required for m6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

• Klíčová slova
• Arabidopsis, HAKAI, N6-adenosine methylation (m6A), VIRILIZER, mRNA methylation, protoxylem,
• MeSH
• adenosin metabolismus   MeSH
• Arabidopsis metabolismus   MeSH
• geneticky modifikované rostliny metabolismus   MeSH
• konzervovaná sekvence MeSH
• messenger RNA metabolismus   MeSH
• methyltransferasy genetika   metabolismus   fyziologie   MeSH
• metylace MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• sekvenční seřazení MeSH
• ubikvitinligasy genetika   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 6-methyladenine mRNA methyltransferase MeSH Prohlížeč
• adenosin MeSH
• messenger RNA MeSH
• methyltransferasy MeSH
• proteiny huseníčku MeSH
• ubikvitinligasy MeSH

With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

• MeSH
• Arabidopsis genetika   MeSH
• cytosol imunologie   metabolismus   MeSH
• fosfotransferasy biosyntéza   genetika   imunologie   MeSH
• mapy interakcí proteinů genetika   imunologie   MeSH
• proteinkinasy biosyntéza   genetika   imunologie   MeSH
• proteiny huseníčku biosyntéza   genetika   imunologie   MeSH
• protilátky aplikace a dávkování   imunologie   MeSH
• regulace genové exprese u rostlin MeSH
• rekombinantní proteiny aplikace a dávkování   imunologie   MeSH
• signální transdukce MeSH
• umlčování genů imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AHP3 protein, Arabidopsis MeSH Prohlížeč
• CKI1 protein, Arabidopsis MeSH Prohlížeč
• fosfotransferasy MeSH
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• protilátky MeSH
• rekombinantní proteiny MeSH

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.

• Klíčová slova
• AHP2, Arabidopsis thaliana, histidine phosphotransfer proteins, multi-step phosphorelay,
• MeSH
• Arabidopsis metabolismus   MeSH
• difrakce rentgenového záření MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfotransferasy chemie   izolace a purifikace   MeSH
• krystalizace MeSH
• proteiny huseníčku chemie   izolace a purifikace   MeSH
• signální transdukce * MeSH
• tranzitní teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AHP2 protein, Arabidopsis MeSH Prohlížeč
• fosfotransferasy MeSH
• proteiny huseníčku MeSH