Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis

The ethylene receptor ETR1 of Arabidopsis contains transmembrane domains responsible for ethylene binding and membrane localization. Sequence analysis does not provide information as to which membrane system of the plant cell ETR1 is localized. Examination by aqueous two-phase partitioning, sucrose density-gradient centrifugation, and immunoelectron microscopy indicates that ETR1 is predominantly localized to the endoplasmic reticulum. Localization of ETR1 showed no change following a cycloheximide chase. Ethylene binding by ETR1 did not affect localization to the endoplasmic reticulum, based upon analysis of plants treated with the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid and by examination of a mutant receptor that does not bind ethylene. Determinants within the amino-terminal half of ETR1 are sufficient for targeting to and retention at the endoplasmic reticulum. These data support a central role of the plant endoplasmic reticulum in hormone perception and signal transduction.     

Cyanide is an adequate agonist of the plant hormone ethylene for studying signalling of sensor kinase ETR1 at the molecular level

The plant hormone ethylene is involved in many developmental processes and responses to environmental stresses in plants. Although the elements of the signalling cascade and the receptors operating the ethylene pathway have been identified, a detailed understanding of the molecular processes related to signal perception and transfer is still lacking. Analysis of these processes using purified proteins in physical, structural and functional studies is complicated by the gaseous character of the plant hormone. In the present study, we show that cyanide, a π-acceptor compound and structural analogue of ethylene, is a suitable substitute for the plant hormone for in vitro studies with purified proteins. Recombinant ethylene receptor protein ETR1 (ethylene-resistant 1) showed high level and selective binding of [(14)C]cyanide in the presence of copper, a known cofactor in ethylene binding. Replacement of Cys(65) in the ethylene-binding domain by serine dramatically reduced binding of radiolabelled cyanide. In contrast with wild-type ETR1, autokinase activity of the receptor is not reduced in the ETR1-C65S mutant upon addition of cyanide. Additionally, protein-protein interaction with the ethylene signalling protein EIN2 (ethylene-insensitive 2) is considerably sustained by cyanide in wild-type ETR1, but is not affected in the mutant. Further evidence for the structural and functional equivalence of ethylene and cyanide is given by the fact that the ethylene-responsive antagonist silver, which is known to allow ligand binding but prevent intrinsic signal transduction, also allows specific binding of cyanide, but shows no effect on autokinase activity and ETR1-EIN2 interaction.     

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.     

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.     

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.     

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into α-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.     

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。     

拟南芥乙烯信号传递途径

植物激素乙烯早在一百多年前就已经被确认,相关的研究使得乙烯广泛地被应用于农业上.一直到十年前第一个植物激素乙烯受体拟南芥ETR1基因被发现之后,人们对于乙烯信号传递的研究并才真正开始有所突破.以遗传学为基础对乙烯反应突变体所做的分析,使得乙烯信号传递已经成为目前植物信号传递领域中被研究得最清楚的信号传递途径之一.该文着重于回顾乙烯信号传递途径上各个元件的发现和确认,以及如何利用遗传学的方法将现有的突变体相关基因构建出目前广为接受的信号传递的遗传模式.最后,该文就目前所知的乙烯信号传递理论及相关研究,做了总结和深入的讨论.     

Control of ethylene-mediated processes in tomato at the level of receptors

The plant hormone ethylene controls many aspects of development and response to the environment. In tomato, ethylene is an essential component of flower senescence, organ abscission, adventitious root initiation, and fruit ripening. Responses to ethylene are also critical for aspects of biotic and abiotic stress responses. Clearly, much of the control of these events occurs at the level of hormone synthesis. However, it is becoming apparent that levels of the ethylene receptors are also highly regulated. The tomato ethylene receptors are encoded by a family of six genes. Levels of expression of these genes are spatially and temporally controlled throughout development. Further, a subset of the receptor genes respond to external stimuli. Genetic and biochemical evidence supports a model in which the ethylene receptors act as negative regulators of downstream responses; in the absence of ethylene, receptors actively suppress expression of ethylene responsive genes. Consistent with this model, a reduction in the overall level of receptor increases ethylene responsiveness of a tissue while higher expression of receptor decreases ethylene sensitivity. Evidence to support this model will be presented.     

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.     

Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1-349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K(d), 117 nM) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K(d), 1.38 μM). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.     

EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.

The Arabidopsis ethylene receptor gene ETR1 and two related genes, ERS1 and ETR2, were identified previously. These three genes encode proteins homologous to the two-component regulators that are widely used for environment sensing in bacteria. Mutations in these genes confer ethylene insensitivity to wild-type plants. Here, we identified two Arabidopsis genes, EIN4 and ERS2, by cross-hybridizing them with ETR2. Sequence analysis showed that they are more closely related to ETR2 than they are to ETR1 or ERS1. EIN4 previously was isolated as a dominant ethylene-insensitive mutant. ERS2 also conferred dominant ethylene insensitivity when certain mutations were introduced into it. Double mutant analysis indicated that ERS2, similar to ETR1, ETR2, ERS1, and EIN4, acts upstream of CTR1. Therefore, EIN4 and ERS2, along with ETR1, ETR2, and ERS1, are members of the ethylene receptor-related gene family of Arabidopsis. RNA expression patterns of members of this gene family suggest that they might have distinct as well as redundant functions in ethylene perception.     

Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1-AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta.