Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis.
TL;DR: The results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.
Abstract: CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.
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TL;DR: Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 3, and 5, and future work needs to focus on identifying substrates ofMAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.
Abstract: Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include crossinhibition, feedback control, and scaffolding Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5 Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways
908 citations
TL;DR: The elucidation of the biochemical mechanisms of ethylene signal transduction and the identification of new components in the ethylene response pathway in Arabidopsis are providing a framework for understanding how all plants sense and respond to ethylene.
901 citations
TL;DR: It is reported that EIN3 protein levels rapidly increase in response to ethylene and this response requires several ethylene-signaling pathway components including the ethylene receptors (ETR1 and EIN4), CTR1, Ein2, EIN5, and Ein6, and the ubiquitin/proteasome pathway.
831 citations
Cites background from "Biochemical and functional analysis..."
...…suggesting that the eth- ylene signaling is not completely blocked in these plants. signaling components are protein kinases (Gamble et al., 1998; Huang et al., 2003; Ouaked et al., 2003), it isFurthermore, the level of EIN3 remains weakly respon- sive to ethylene in ctr1-1 mutants, although…...
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TL;DR: A surprisingly large number of genes encoding MAPK pathway components have been uncovered by analysing model plant genomes, suggesting that MAPK cascades are abundant players of signal transduction.
616 citations
TL;DR: This review integrates knowledge from the model plant Arabidopsis and other plant species and focuses on key aspects of recent research on regulatory networks controlling ethylene synthesis and its role in flower development and fruit ripening.
Abstract: Ethylene regulates many aspects of the plant life cycle, including seed germination, root initiation, flower development, fruit ripening, senescence, and responses to biotic and abiotic stresses. It thus plays a key role in responses to the environment that have a direct bearing on a plant's fitness for adaptation and reproduction. In recent years, there have been major advances in our understanding of the molecular mechanisms regulating ethylene synthesis and action. Screening for mutants of the triple response phenotype of etiolated Arabidopsis seedlings, together with map-based cloning and candidate gene characterization of natural mutants from other plant species, has led to the identification of many new genes for ethylene biosynthesis, signal transduction, and response pathways. The simple chemical nature of ethylene contrasts with its regulatory complexity. This is illustrated by the multiplicity of genes encoding the key ethylene biosynthesis enzymes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, multiple ethylene receptors and signal transduction components, and the complexity of regulatory steps involving signalling relays and control of mRNA and protein synthesis and turnover. In addition, there are extensive interactions with other hormones. This review integrates knowledge from the model plant Arabidopsis and other plant species and focuses on key aspects of recent research on regulatory networks controlling ethylene synthesis and its role in flower development and fruit ripening.
606 citations
Cites background from "Biochemical and functional analysis..."
...Mutation of the conserved N-terminal CN motif in both the Arabidopsis CTR1 (Gao et al., 2003; Huang et al., 2003) and tomato CTR3 (Zhong et al....
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...Mutation of the conserved N-terminal CN motif in both the Arabidopsis CTR1 (Gao et al., 2003; Huang et al., 2003) and tomato CTR3 (Zhong et al., 2008a) can disrupt their interaction with the receptors and results in accumulation of free CTR proteins in the cytosol....
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References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
Book•
01 Jan 1988
TL;DR: A second edition of Antibodies: A Laboratory Manual is being published in September 2013, Revised, extended and updated by Edward Greenfield of the Dana-Farber Cancer Center, the material has been recast with extensive new information and new chapters have been added.
Abstract: ince its publication in 1988, Antibodies: A Laboratory Manual, by Harlow and Lane, has become a classic, an essential resource for molecular biology, immunology, and cell culture labs. In order to keep the book in print, Cold Spring Harbor Laboratory Press eventually produced the paperback edition currently available for sale. Now, after 25 years, a second edition is being published in September 2013. Revised, extended and updated by Edward Greenfield of the Dana-Farber Cancer Center, the material has been recast with extensive new information and new chapters have been added. The new edition provides clear, authoritative, current and up-to-date protocols with background information and troubleshooting advice. The book is an invaluable resource for all those engaged in antibody research and development.
22,254 citations
TL;DR: A novel genetic system to study protein-protein interactions between two proteins by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae, which may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Abstract: Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
6,529 citations
TL;DR: This work has developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis that is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction.
Abstract: The polymerase chain reaction (PCR) has revolutionised the rapid analysis of mammalian genomic DNA (1). However, PCR is less useful in the analysis of plant DNA due to the difficulties in extracting nucleic acids from limited amounts of plant tissue. We have developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis. The method is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction. Thus it is possible to complete an extraction within 15 minutes without handling any hazardous organic solvents. Samples for PCR analysis (usually leaf tissue) are collected using the lid of a sterile Eppendorf tube to pinch out a disc of material into the tube. This ensures uniform sample size and also reduces the possibilities of contamination arising from handling the tissue. DNA is extracted as follows: The tissue is macerated (using disposable grinders from Bel-art Products: Scienceware, Pequannock, NJ, 07440 USA. catalog no 992) in the original Eppendorf tube at room temperature, without buffer, for 15 seconds. 400 yl of extraction buffer (200 mM Tris HC1 pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the sample vortexed for 5 seconds. This mixture can then be left at room temperature until all the samples have been extracted (> 1 hour). The extracts are centrifuged at 13,000 rpm for 1 minute and 300 y\\ of the supernatant transferred to a fresh Eppendorf tube. This supernatant is mixed with 300 /il isopropanol and left at room temperature for 2 minutes. Following centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum dried and dissolved in 100 /xl 1XTE. This DNA is stable at 4°C for greater than one year. 2.5 yX of this sample is sufficient for a standard 50 y\\ PCR (Figure 1). When older tissue is used this may be increased to 25 /xl without any deleterious effect on the PCR. Using this protocol we have found it possible to process hundreds of individual samples in a single working day.
2,482 citations