Arabidopsis ETR1 and ERS1 differentially repress the ethylene response in combination with other ethylene receptor genes

The ethylene response is negatively regulated by a family of five ethylene receptor genes in Arabidopsis (Arabidopsis thaliana). The five members of the ethylene receptor family can physically interact and form complexes, which implies that cooperativity for signaling may exist among the receptors. The ethylene receptor gene mutations etr1-1((C65Y))(for ethylene response1-1), ers1-1((I62P)) (for ethylene response sensor1-1), and ers1(C65Y) are dominant, and each confers ethylene insensitivity. In this study, the repression of the ethylene response by these dominant mutant receptor genes was examined in receptor-defective mutants to investigate the functional significance of receptor cooperativity in ethylene signaling. We showed that etr1-1((C65Y)), but not ers1-1((I62P)), substantially repressed various ethylene responses independent of other receptor genes. In contrast, wild-type receptor genes differentially supported the repression of ethylene responses by ers1-1((I62P)); ETR1 and ETHYLENE INSENSITIVE4 (EIN4) supported ers1-1((I62P)) functions to a greater extent than did ERS2, ETR2, and ERS1. The lack of both ETR1 and EIN4 almost abolished the repression of ethylene responses by ers1(C65Y), which implied that ETR1 and EIN4 have synergistic effects on ers1(C65Y) functions. Our data indicated that a dominant ethylene-insensitive receptor differentially repressed ethylene responses when coupled with a wild-type ethylene receptor, which supported the hypothesis that the formation of a variety of receptor complexes may facilitate differential receptor signal output, by which ethylene responses can be repressed to different extents. We hypothesize that plants can respond to a broad ethylene concentration range and exhibit tissue-specific ethylene responsiveness with differential cooperation of the multiple ethylene receptors.     

Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), ethylene is perceived by a receptor family consisting of five members. Subfamily 1 members ETHYLENE RESPONSE1 (ETR1) and ETHYLENE RESPONSE SENSOR1 (ERS1) have histidine kinase activity, unlike the subfamily 2 members ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4), which lack amino acid residues critical for this enzymatic activity. To resolve the role of histidine kinase activity in signaling by the receptors, we transformed an etr1-9;ers1-3 double mutant with wild-type and kinase-inactive versions of the receptor ETR1. Both wild-type and kinase-inactive ETR1 rescue the constitutive ethylene-response phenotype of etr1-9;ers1-3, restoring normal growth to the mutant in air. However, the lines carrying kinase-inactive ETR1 exhibit reduced sensitivity to ethylene based on several growth response assays. Microarray and real-time polymerase chain reaction analyses of gene expression support a role for histidine kinase activity in eliciting the ethylene response. In addition, protein levels of the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which physically associates with the ethylene receptor ETR1, are less responsive to ethylene in lines containing kinase-inactive ETR1. These data indicate that the histidine kinase activity of ETR1 is not required for but plays a modulating role in the regulation of ethylene responses. Models for how enzymatic and nonenzymatic regulation may facilitate signaling from the ethylene receptors are discussed.     

Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis

Ethylene, a regulator of plant growth and development, is perceived by specific receptors that act as negative regulators of the ethylene response. Five ethylene receptors, i.e., ETR1, ERS1, EIN4, ETR2, and ERS2, are present in Arabidopsis and dominant negative mutants of each that confer ethylene insensitivity have been reported. In contrast, maize contains just two types of ethylene receptors: ZmERS1, encoded by ZmERS1a and ZmERS1b, and ZmETR2, encoded by ZmETR2a and ZmETR2b. In this study, we introduced a Cys to Tyr mutation in the transmembrane domain of ZmERS1b and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and Arabidopsis expressing Zmers1b or Zmetr2b were larger and exhibited a delay in leaf senescence characteristic of ethylene insensitive Arabidopsis mutants. Zmers1b and Zmetr2b were dominant and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. These results suggest that Cys65 in maize ZmERS1b and ZmETR2b plays the same role that it does in Arabidopsis receptors. Moreover, the results demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.     

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors <Emphasis Type="Italic">ETR1</Emphasis> and <Emphasis Type="Italic">ERS1</Emphasis>

Background  

The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations.     

The Relationship between Ethylene Binding and Dominant Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene Receptor

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.     

Heteromeric interactions among ethylene receptors mediate signaling in Arabidopsis

The gaseous hormone ethylene is perceived in Arabidopsis by a five member receptor family that consists of the subfamily 1 receptors ETR1 and ERS1 and the subfamily 2 receptors ETR2, ERS2, and EIN4. Previous work has demonstrated that the basic functional unit for the ethylene receptor, ETR1, is a disulfide-linked homodimer. We demonstrate here that ethylene receptors isolated from Arabidopsis also interact with each other through noncovalent interactions. Evidence that ETR1 associates with other ethylene receptors was obtained by co-purification of ETR1 with tagged versions of ERS1, ETR2, ERS2, and EIN4 from Arabidopsis membrane extracts. ETR1 preferentially associated with the subfamily 2 receptors compared with the subfamily 1 receptor ERS1, but ethylene treatment affected the interactions and relative composition of the receptor complexes. When transgenically expressed in yeast, ETR1 and ERS2 can form disulfide-linked heterodimers. In plant extracts, however, the association of ETR1 and ERS2 can be largely disrupted by treatment with SDS, supporting a higher order noncovalent interaction between the receptors. Yeast two-hybrid analysis demonstrated that the receptor GAF domains are capable of mediating heteromeric receptor interactions. Kinetic analysis of ethylene-insensitive mutants of ETR1 is consistent with their dominance being due in part to an ability to associate with other ethylene receptors. These data suggest that the ethylene receptors exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked chemoreceptors of bacteria and that interactions among receptors contribute to ethylene signal output.     

Mutational analysis of the ethylene receptor ETR1. Role of the histidine kinase domain in dominant ethylene insensitivity

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The N-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The C-terminal half of the polypeptide contains domains with homology to histidine (His) kinases and response regulators, signaling motifs originally identified in bacteria. The role of the His kinase domain in ethylene signaling was examined in planta. For this purpose, site-directed mutations were introduced into the full-length wild-type ETR1 gene and into etr1-1, a mutant allele that confers dominant ethylene insensitivity on plants. The mutant forms of the receptor were expressed in Arabidopsis and the transgenic plants characterized for their ethylene responses. A mutation that eliminated His kinase activity did not affect the ability of etr1-1 to confer ethylene insensitivity. A truncated version of etr1-1 that lacks the His kinase domain also conferred ethylene insensitivity. Possible mechanisms by which a truncated version of etr1-1 could exert dominance are discussed.     

植物乙烯信号转导研究进展

过去10年,对模式植物拟南芥的分子遗传学研究建立了植物乙烯信号转导线性模型.乙烯结合到受体上,经一条MAPK级联反应和转录级联途径将信号转导而产生乙烯反应.拟南芥乙烯受体家族由5个成员构成,ETR1、ERS1、ETR2、ERS2和EIN4.乙烯受体包括三个结构域:乙烯结合结构域、组氨酸激酶结构域和反应调控结构域.乙烯受体定位于内质网,与CTR1协同负调控乙烯反应.ENI2、EIN3/EIL、ERF1依次位于CTR1下游,正调控乙烯反应.EIN3属于转录激活因子调控蛋白家族,受转录后调控.乙烯稳定EIN3结构,EBF1/EBF2促进EIN3分解.ERF1是转录调控因子家族成员之一,是EIN3/EIL的直接作用目标.     

Ethylene perception by the ERS1 protein in Arabidopsis

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.     

乙烯的生物合成与信号传递

乙烯是气体植物激素, 它在植物的生长发育过程中有很多作用。所以了解乙烯的生物合成及其信号转导是非常重要的。二十年来, 通过筛选有异于正常三重反应的突变体, 人们发现了乙烯信号转导的粗略轮廓。在拟南芥中, 有5个受体蛋白感受乙烯, ETR1、ERS1、ETR2、ERS2、EIN4。它们表现出功能冗余, 是乙烯信号的负调控因子, 在植物体内以二聚体的形式存在。ETR1的N端与乙烯结合时需要 铜离子(Ⅰ)的参与。尽管已经发现ETR1有组氨酸激酶活性, 而其它受体有丝氨酸/苏氨酸激酶活性, 但受体参与乙烯信号转导的机制还不是很清楚。受体与Raf类蛋白激酶CTR1相互作用, CTR1是乙烯反应的负调控因子。CTR1蛋白失活使EIN2蛋白活化。EIN2的N端是跨膜结构域, 与Nramp家族金属离子转运蛋白的跨膜结构域类似。EIN2的C端是一个新的未知结构域, 与乙烯信号途径的下游组分相互作用。EIN3位于EIN2的下游, EIN3和EILs诱导ERF1和其它转录因子的表达, 这些转录因子依次激活乙烯反应目的基因的表达, 表现出乙烯的反应。EIN3受到蛋白酶体介导的蛋白降解途径的调节。由于乙烯是一种多功能的植物激素, 其信号途径与其它信号途径有多重的交叉。     

乙烯信号转导的分子机制

气态植物激素乙烯在植物生长发育和应对生物及非生物胁迫过程中起着重要作用。在过去的十几年中, 对模式植物拟南芥的分子遗传研究已建立从信号感知到转录调控的乙烯信号转导线性模型。拟南芥共有5个乙烯受体ETR1、ERS1、ETR2、ERS2和EIN4, 目前已知ETR1定位在内质网上, 与类似于Raf的蛋白激酶CTR1协同负调控乙烯反应。EIN2和EIN3/EILs位于CTR1下游, 正调控乙烯反应。两个F-box蛋白EBF1和EBF2通过泛素/26S蛋白体降解途径调控EIN3的稳定性。5’→3’的外切核酸酶EIN5通过启动EBF1和EBF2 mRNA的降解, 拮抗EBF1和EBF2对EIN3的负反馈调控。目前对于乙烯信号转导途径关键组分的生化功能和乙烯下游反应途径的了解甚少, 乙烯信号转导途径与其它途径之间还存在着广泛的交叉反应, 这些问题的解决将大大增加我们对乙烯信号转导途径的了解。     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

Ethylene Receptors Function as Components of High-Molecular-Mass Protein Complexes in Arabidopsis

Background

The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4.

Methodology/Principal Findings

Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding.

Conclusions/Significance

These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.     

Myo-Inositol-Dependent Sodium Uptake in Ice Plant

The phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. Much of the developmental regulation of ethylene responses in tomato (Lycopersicon esculentum) occurs at the level of hormone sensitivity. In an effort to understand the regulation of ethylene responses, we isolated and characterized tomato genes with sequence similarity to the Arabidopsis ETR1 (ethylene response 1) ethylene receptor. Previously, we isolated three genes that exhibit high similarity to ETR1 and to each other. Here we report the isolation of two additional genes, LeETR4 and LeETR5, that are only 42% and 40% identical to ETR1, respectively. Although the amino acids known to be involved in ethylene binding are conserved, LeETR5 lacks the histidine within the kinase domain that is predicted to be phosphorylated. This suggests that histidine kinase activity is not necessary for an ethylene response, because mutated forms of both LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic Arabidopsis plants. Expression analysis indicates that LeETR4 accounts for most of the putative ethylene-receptor mRNA present in reproductive tissues, but, like LeETR5, it is less abundant in vegetative tissues. Taken together, ethylene perception in tomato is potentially quite complex, with at least five structurally divergent, putative receptor family members exhibiting significant variation in expression levels throughout development.     

Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments

A cDNA from deep water rice treated with ethylene, encoding an ethylene receptor homologous to Arabidopsis thaliana ETR2 and EIN4, was isolated using differential display and RACE techniques. The cDNA (2880 bp), corresponding to the Os-ERL1 gene (Oryza sativa ETHYLENE RESPONSE 2 like 1; GenBank accession number AB107219), contained an open reading frame of 2289 bp coding for 763 amino acids. The protein Os-ERL1 has 50% and 52% similarity to Arabidopsis ETR2 and EIN4, respectively. The Os-ERL1 gene was up-regulated by flooding, and by treatment with ethylene and gibberellin. These results suggest that deep water rice responds to flooding by increasing the number of its ethylene receptors.