ETR1 Receptor Research Articles

Arabidopsis CPR5 plays a role in regulating nucleocytoplasmic transport of mRNAs in ethylene signaling pathway.

Arabidopsis CPR5 is involved in regulation of ethylene signaling via two different ways: interacting with the ETR1 N-terminal domains, and controlling nucleocytoplasmic transport of ethylene-related mRNAs. The ETR1 receptor plays a predominant role in ethylene signaling in Arabidopsis thaliana. Previous studies showed that both RTE1 and CPR5 can directly bind to the ETR1 receptor and regulate ethylene signaling. RTE1 was suggested to promote the ETR1 receptor signaling by influencing its conformation, but little is known about the regulatory mechanism of CPR5 in ethylene signaling. In this study, we presented the data showing that both RTE1 and CPR5 bound to the N-terminal domains of ETR1, and regulated ethylene signaling via the ethylene receptor. On the other hand, the research provided evidence indicating that CPR5 could act as a nucleoporin to regulate the ethylene-related mRNAs export out of the nucleus, while RTE1 or its homolog (RTH) had no effect on the nucleocytoplasmic transport of mRNAs. Nuclear qRT-PCR analysis and poly(A)-mRNA in situ hybridization showed that defect of CPR5 restricted nucleocytoplasmic transport of mRNAs. These results advance our understanding of the regulatory mechanism of CPR5 in ethylene signaling.

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.

The ethylene receptor ETR1 is required for Fusarium oxysporum pathogenicity

Fusarium oxysporum is a ubiquitous vascular wilt plant pathogen causing severe yield losses in a wide range of economically important crops. In this study, the interaction between Fusarium oxysporum f. sp. raphani and Arabidopsis thaliana plants impaired in the salicylate (SA), jasmonate (JA) and ethylene (ET) defence signalling pathways was investigated to better understand the nature of this plant–microbe interaction. The in planta bioassays revealed a key role for the ETR1 receptor as the etr1‐1 mutant plants exhibited statistically less Fusarium wilt symptoms compared to the other mutant and Col‐0 plants. Quantitative polymerase chain reaction (qPCR) analysis associated the decrease in symptom severity shown in etr1‐1 plants with reduced vascular growth of the pathogen, suggesting the activation of defence mechanisms in etr1‐1 plants against F. oxysporum. Furthermore, the early activation and increased accumulation of the SA‐responsive PR1, PR2 and PR5 genes in the etr1‐1 plants, in contrast to the Col‐0 plants that showed higher transcript levels of the JA/ET‐responsive PR3, PR4 and PDF1.2 genes after F. oxysporum inoculation, can lead to speculation that F. oxysporum hijacks ETR1‐mediated ethylene signalling to promote disease development in plants.

The effects of Group 11 transition metals, including gold, on ethylene binding to the ETR1 receptor and growth of Arabidopsis thaliana

It has been previously shown that Cu(I) and the ethylene response antagonist, Ag(I), support ethylene binding to exogenously expressed ETR1 ethylene receptors. Both are Group 11 transition metals that also include gold. We compared the effects of gold ions with those of Cu(I) and Ag(I) on ethylene binding in exogenously expressed ETR1 receptors and on ethylene growth responses in etiolated Arabidopsis seedlings. We find that gold ions also support ethylene binding but, unlike Ag(I), do not block ethylene action on plants. Instead, like Cu(I), gold ions affect seedlings independently of ethylene signaling.

Identification of Important Regions for Ethylene Binding and Signaling in the Transmembrane Domain of the ETR1 Ethylene Receptor of Arabidopsis

The ethylene binding domain (EBD) of the Arabidopsis thaliana ETR1 receptor is modeled as three membrane-spanning helices. We surveyed ethylene binding activity in different kingdoms and performed a bioinformatic analysis of the EBD. Ethylene binding is confined to land plants, Chara, and a group of cyanobacteria but is largely absent in other organisms, consistent with our finding that EBD-like sequences are overrepresented among plant and cyanobacterial species. We made amino acid substitutions in 37 partially or completely conserved residues of the EBD and assayed their effects on ethylene binding and signaling. Mutations primarily in residues in Helices I and II midregions eliminated ethylene binding and conferred constitutive signaling, consistent with the inverse-agonist model of ethylene receptor signaling and indicating that these residues define the ethylene binding pocket. The largest class of mutations, clustered near the cytoplasmic ends of Helices I and III, gave normal ethylene binding activity yet still conferred constitutive signaling. Therefore, these residues may play a role in turning off the signal transmitter domain of the receptor. By contrast, only two mutations were loss of function with respect to signaling. These findings yield insight into the structure and function of the EBD and suggest a conserved role of the EBD as a negative regulator of the signal transmitter domain.

Ethylene-Stimulated Nutations Do Not Require ETR1 Receptor Histidine Kinase Activity

Ethylene influences the growth and development of plants through the action of receptors that have homology to bacterial two-component receptors. In bacteria these receptors function via autophosphorylation of a His residue in the kinase domain followed by phosphotransfer to a conserved Asp residue in a response regulator protein. In Arabidopsis, two of the five receptor isoforms are capable of His kinase activity. However, the role of His kinase activity and phosphotransfer is unclear in ethylene signaling. A previous study showed that ethylene stimulates nutations of the hypocotyl in etiolated Arabidopsis seedlings that are dependent on the ETR1 receptor isoform. The ETR1 receptor is the only isoform in Arabidopsis that contains both a functional His kinase domain and a receiver domain for phosphotransfer. Therefore, we examined the role that ETR1 His kinase activity and phosphotransfer plays in ethylene-stimulated nutations.

Ethylene stimulates nutations that are dependent on the ETR1 receptor.

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.

Receptor Signal Output Mediated by the ETR1 N Terminus Is Primarily Subfamily I Receptor Dependent

etr1-1 is a dominant ethylene receptor gene in Arabidopsis (Arabidopsis thaliana) and confers ethylene insensitivity. The truncated etr1-1(1-349) protein is capable of repressing ethylene responses, whereas etr1(1-349) is not, lending support to a hypothesis that the dominant etr1-1(1-349) could convert wild-type receptors to an ethylene-insensitive state. Assuming that etr1-1(1-349) and etr1(1-349) would share the same signaling mechanism, we hypothesize that the etr1(1-349) protein is capable of repressing ethylene responses when not bound with ethylene. In this study, we show that both etr1(1-349) and etr1-1(1-349) are capable of receptor signal output, which is primarily dependent on subfamily I receptors. The etr1(1-349) and etr1-1(1-349) clones were individually transformed to mutants and the resulting phenotypes were scored. Each of those transgenes restored the rosette growth and flower fertility of etr1-7 ers1-2 to a similar extent. In contrast, neither etr1(1-349) nor etr1-1(1-349) was capable of signal output in etr1-7 ers1-3. The ERS1 transcript was detectable in ers1-2 but not in ers1-3, implying that ETR1 N-terminal signaling is subfamily I dependent. Loss of the subfamily II receptor genes did not perturb etr1-1(1-349)-mediated ethylene insensitivity. Possible roles of subfamily I receptors and disulfide linkages in ETR1 receptor signal output mediated through the N terminus are discussed.

Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.

In Arabidopsis, ethylene is perceived by a receptor family consisting of five members, one of these being ETR1. The N-terminal half of ETR1 functions as a signal input domain. The C-terminal region of ETR1, consisting of a His kinase domain and a putative receiver domain, is likely to function in signal output. The role of the proposed signal output region in ethylene signaling was examined in planta. For this purpose, the ability of mutant versions of ETR1 to rescue the constitutive ethylene-response phenotype of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant line was examined. A truncated version of ETR1 that lacks both the His kinase domain and the receiver domain failed to rescue the triple mutant phenotype. A truncated ETR1 receptor that lacks only the receiver domain restored normal growth to the triple mutant in air, but the transgenic seedlings displayed hypersensitivity to low doses of ethylene. A mutation that eliminated His kinase activity had a modest effect upon the ability of the receptor to repress ethylene responses in air. These results demonstrate that the His kinase domain plays a role in the repression of ethylene responses. The potential roles of the receiver domain and His kinase activity in ethylene signaling are discussed.

Temporary Relationship Between C2 H4 Evolution and C2 H4 Recptor Genes

`Granny Smith' apples were stored at 1 °C, 5 °C and 20 °C, then treated at the preclimacteric stage with 2 ppm MCP, various O2 concentrations, and MCP + low O2. All the treatments greatly retarded the onset of the C2 H4 climacteric, and hence ripening. MCP + low O2 was much more effective than were the applications of MCP and low O2 singly. Even at 20 °C, 4.04 kPa O2 inhibited the rise in C2 H4 evolution for 145 d. Neither low O2 nor MCP inhibited the System 1 C2 H4 evolution. The suppression of the climacteric rise in C2 H4 evolution was accompanied by a strong inhibition of the accumulation of ERS1 C2 H4 receptor and ACS transcripts. On the other hand, ETR1 receptor was constitutively expressed. When climacteric fruits were treated with MCP, and with low O2 + MCP the rate of C2 H4 evolution decreased sharply. This occurred simultaneously with a decrease in ERS1 mRNA. Moreover, the decrease in ERS1 mRNA paralleled the decrease in C2 H4 evolution. The data thus indicate that the initiation and sustainment of the C2 H4 climacteric requires the presence of functional C2 H4 receptors. The expression of ETR2 and ERS2 is also under investigation.

High-level expression of the Arabidopsis thaliana ethylene receptor protein ETR1 in Escherichia coli and purification of the recombinant protein

Ethylene responses in plants are mediated by a small family of membrane integral receptors including the ETR1 gene product which are similar to the two-component bacterial histidine kinase regulators. Detailed biochemical and structural analysis of the ethylene-receptor family was hampered by the scarce amount of pure protein. Here, we report the construction, expression, and single-step purification of the ETR1 receptor protein from Arabidopsis thaliana in a bacterial expression system. The DNA fragment encoding the mature ETR1 receptor protein was subcloned into the pET15b expression vector and highly expressed in derivatives C41(DE3) and C43(DE3) of the Escherichia coli strain BL21(DE3). The recombinant protein was solubilised from the bacterial cells using mild non-denaturing detergents and purified to homogeneity by Ni–NTA affinity chromatography, yielding approximately 2–3 mg pure protein per litre of cells. The molecular mass of the purified protein was estimated to be 78 kDa by SDS–PAGE. The expression and purification of recombinant ETR1 reported here provide a basis for detailed functional and structural studies of the receptor protein, which might help to understand signal perception and signal transduction of the phytohormone ethylene on the molecular level.

A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.

The ETR1 receptor from Arabidopsis binds the gaseous hormone ethylene. A copper ion associated with the ethylene-binding domain is required for high-affinity ethylene-binding activity. A missense mutation in the domain that renders the plant insensitive to ethylene eliminates both ethylene binding and the interaction of copper with the receptor. A sequence from the genome of the cyanobacterium Synechocystis sp. strain 6803 that shows homology to the ethylene-binding domain of ETR1 encodes a functional ethylene-binding protein. On the basis of sequence conservation between the Arabidopsis and the cyanobacterial ethylene-binding domains and on in vitro mutagenesis of ETR1, a structural model for this copper-based ethylene sensor domain is presented.