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Review articles Ethylene hormone receptor action in Arabidopsis Caren Chang* and Ruth Stadler Summary Small gaseous molecules play important roles in biological signaling in both animal and plant physiology. The hydrocarbon gas ethylene has long been known to regulate diverse aspects of plant growth and development, including fruit ripening, leaf senescence and flower abscission. Recent progress has been made toward identifying components involved in ethylene signal transduction in the plant Arabidopsis thaliana. Ethylene is perceived by five receptors that have similarity to twocomponent signaling proteins. The hydrophobic aminoterminus of the receptors binds ethylene, and mutations in this domain both prevent ethylene binding and confer ethylene insensitivity to the plant; the carboxyl-terminal portion of the receptors has similarity to bacterial histidine protein kinases. Genetic data suggest a model in which ethylene binding inhibits receptor signaling, yet precisely how these receptors function is unclear. Two of the receptors have been found to associate with a negative regulator of ethylene responses called CTR1, which appears to be a mitogen-activated protein kinase (MAPK) kinase kinase. BioEssays 23:619±627, 2001. ß 2001 John Wiley & Sons, Inc. Department of Cell Biology and Molecular Genetics, University of Maryland. Funding agencies: NRI Competitive Grants Program/USDA (Grant No:98-35304-67-95), Department of Energy (Grant No 02-99ER20329), a German Science Society Fellowship to R. Stadler and the Maryland Agricultural Experiment Station. *Correspondence to: Dr. Caren Chang, Department of Cell Biology and Molecular Genetics, HJ Patterson Hall, University of Maryland, College Park, MD 20782 USA. E-mail: [email protected] Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AIN, ACC-INSENSITIVE; CTR, CONSTITUTIVE TRIPLE RESPONSE; EIL, EIN3-LIKE; EIN, ETHYLENE INSENSITIVE; ERE, ETHYLENE RESPONSE ELEMENT; EREBP, ETHYLENE RESPONSE ELEMENT-BINDING PROTEIN; ERF, ETHYLENE RESPONSE FACTOR; ERS, ETHYLENE RESPONSE SENSOR; ETR, ETHYLENE RESISTANT (now considered as ETHYLENE RECEPTOR); GAF, cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA; HPK, histidine protein kinase; HPt, histidine-containing phosphotransfer; MAPK, mitogenactivated protein kinase; RAN, RESPONSIVE TO ANTAGONIST. BioEssays 23:619±627, ß 2001 John Wiley & Sons, Inc. Introduction Both plants and animals utilize hormones to communicate between cells and organs. These messenger molecules enable the organism to develop and respond to internal and external cues in a coordinated manner. Interestingly, some signal molecules are gaseous-nitric oxide is the leading example in mammals. In plants, the premier example of a gaseous signal molecule is the simple hydrocarbon ethylene (C2H4). Considered one of the five classic plant hormones, ethylene has long been known to regulate a diverse array of physiological and developmental processes. The best known effect of ethylene is the promotion of fruit ripening.(1) Other notable processes that are regulated by ethylene include seed germination, senescence, and responses to stress factors such as flooding, wounding or pathogen attack.(1) The molecular basis of plant hormone signaling is a complex topic that has only begun to be unraveled. Insight into the ethylene-response pathway has been mainly provided by molecular genetic studies in Arabidopsis thaliana. Dissection of the ethylene-response pathway began with the isolation of ethylene-response mutants, using genetic screens that are based on the ``triple response'' of ethylene-treated seedlings.(2,3) The triple response is a striking morphology adopted by germinating seedlings exposed to ethylene in the dark. In Arabidopsis, the triple response consists of shortening and thickening of the hypocotyl and root, exaggeration of the apical hook curvature and proliferation of root hairs (Fig. 1) .(2,3) This altered morphology is thought to facilitate seedling emergence from the soil,(4) as ethylene biosynthesis is triggered by mechanical stress encountered during germination.(5) In the laboratory, the triple response can be induced in germinating seedlings by treatment with either ethylene gas or its chemical precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (which is taken up by the seedlings and converted to ethylene by ACC oxidase). Due to the simplicity of the assay, nearly all of the ethylene-response mutants known today were identified on the basis of triple response defects. ethylene-resistant (etr ), ethylene-insensitive (ein) and ACC-insensitive (ain) mutants show little or no triple response in the presence of high levels of ethylene or ACC(6,7) (Fig. 1). Constitutive response mutants (ctr) display the triple response even in the absence of ethylene (Fig. 1). The order of action for these loci has been established by epistasis analysis, and many of the corresponding genes have been cloned.(6,7) This has uncovered the BioEssays 23.7 619 Review articles Figure 1. Arabidopsis seedlings grown in the dark in the absence (left) or presence (right) of 10 ml per liter ethylene. The wild-type (WT) develops the typical triple response phenotype in the presence of ethylene: a short and thick hypocotyl with an exaggerated apical hook, and a short and thick root. The constitutive ethylene-response mutant ctr1-1 develops the triple response phenotype even in the absence of ethylene, whereas the ethylene-insensitive mutant etr1-1 shows no triple response even in the presence of ethylene. framework of the ethylene-response pathway; the pathway starts with ethylene perception by a family of receptors and progresses to the regulation of ethylene-response genes (Fig. 2). Recent biochemical and genetic studies have defined the pathway further. This review will focus on the ethylene receptors, highlighting findings concerning ethylene receptor action, their unique properties and some of the questions currently under investigation. Overview of the ethylene-response pathway In Arabidopsis, the molecular cloning of genes involved in ethylene signaling has revealed a variety of signaling modules in the ethylene-response pathway (Fig. 2). The first Arabidopsis ethylene-response mutant that was described was etr1-1.(2) The ETR1 gene(8) was found to encode a protein having striking similarity to receptor histidine kinases of the two-component signaling system.(9) Prior to this, it had been thought that the two-component signaling mechanism was exclusive to bacteria. Since then, a number of two-component homologs have been uncovered in eukaryotes.(10±12) Most recently, it was discovered that a two-component histidine kinase in Arabidopsis is the receptor for the plant hormone cytokinin.(13) Another histidine kinase in Arabidopsis appears to function as an osmosensor.(14) Two-component pathways also control osmoregulation and stress responses in yeast, 620 BioEssays 23.7 Figure 2. Schematic diagram of the ethylene signal transduction pathway in Arabidopsis. Five ethylene receptors (ETR1, ERS1, ETR2, EIN4 and ERS2) relay the ethylene signal to the CTR1 protein kinase, which is a negative regulator of ethylene responses. CTR1 has similarity to the Raf family of MAPKKKs, and is therefore presumed to be the first component of a MAP kinase module (which is indicated by ``?''). EIN2, which acts downstream of CTR1, has an integral membrane domain with similarity to the Nramp family of metal-ion transporters. The specific membrane locations of the ethylene receptors and EIN2 are currently unknown. Downstream of EIN2, a novel transcription factor called EIN3 promotes transcription of ERF1, which encodes a member of the EREBP family of transcription factors. ERF1, in turn, binds to the GCC-box promoter element to activate transcription of specific ethylene-response genes. With the exception of EIN3's activation of ERF1 transcription, the solid arrows in the diagram represent undefined steps in the pathway. and are involved in Dictyostelium and Neurospora development,(12) but have not been found in animal systems. The ETR1 protein was suspected to be an ethylene receptor based on initial genetic and physiological data.(2,8) The subsequent demonstration that ETR1 reversibly binds Review articles ethylene provided strong evidence that ETR1 is indeed an ethylene receptor.(15) Currently, ethylene perception in Arabidopsis is thought to involve not only ETR1, but four ETR1related genes (ERS1, ETR2, EIN4 and ERS2).(16±18) In recent years, substantial progress has been made concerning the structure and function of the ethylene receptors. Diverse plants such as tomato, muskmelon and carnation contain similar families of ethylene receptor genes, suggesting that the mechanism of ethylene signaling is conserved in flowering plants.(10) Downstream of the receptors, the next known component is the protein kinase CTR1. Loss-of-function ctr1 mutants exhibit pleiotropic constitutive ethylene responses, and thus CTR1 is thought to be a negative regulator of ethylene responses.(19) The CTR1 protein kinase domain has high sequence similarity to the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs), suggesting that CTR1 may act in a MAPK phosphorylation cascade. In animals, MAPKKKs are serine/threonine protein kinases, which are activated by growth factors and mitotic signals. MAPKKKs phosphorylate MAPKKs, which in turn phosphorylate MAPKs, which regulate transcription factors. A number of MAPKKK and MAPKK genes have been isolated in plants; however, it is not known whether any of these are involved in ethylene signaling.(20) Regulation of a MAPKKK by two-component histidine kinases appears to be rare; no animal MAP kinase cascades are known to be regulated by two-component proteins, and only two pathways (both in yeast) have been found to contain both a two-component system and a MAP kinase cascade.(21±23) EIN2, which acts downstream of CTR1, is one of the central players in the ethylene-response pathway. Loss-of-function ein2 mutants are highly ethylene-insensitive for all known ethylene responses,(3) and also appear to be defective in their response to several other hormones.(24) The function of the large EIN2 protein is difficult to predict. The aminoterminal portion of EIN2 consists of twelve putative membrane-spanning regions with similarity to the Nramp family of metal ion carriers.(24) Ion transport activity, however, has not been detected for EIN2, and the subcellular localization of EIN2 is unknown. The novel carboxyl-terminal portion is predicted to be a coiled coil, suggesting a role in proteinprotein interactions. Overexpression of truncated or wild-type EIN2 in Arabidopsis indicates that EIN2 is an ethyleneresponse activator with an autoinhibitory amino-terminal domain.(24) A common destination of cellular signaling pathways is the nucleus, where specific transcription factors control gene expression. Two types of ethylene-responsive transcriptional regulators have been identified: the family of ETHYLENE INSENSITIVE3 (EIN3) (and EIN3-LIKE (EIL) proteins)(25,26) and the family of ETHYLENE RESPONSE ELEMENTBINDING PROTEINS (EREBPs).(27±29) Several members of the EIN3/EIL family have been shown to bind a conserved cis- element in the promoter of an EREBP-encoding gene called ETHYLENE RESPONSE FACTOR1 (ERF1).(26) EIN3 activates transcription of ERF1, and ERF1 in turn binds to a cisacting sequence known as the GCC-box located in the promoters of secondary target genes, such as those encoding basic chitinase and defensin PDF1.2.(26,30) ERF1 is thought to be a transcriptional activator, since overexpression of ERF1 activates many ethylene responses. Thus, a signal amplification cascade exists at the transcriptional level to activate ethylene-response genes (Fig. 2).(6) Structurally diverse ``two-component'' ethylene receptors The two-component system is a signaling mechanism used primarily by bacteria to respond to a broad array of environmental signals.(9) Escherichia coli alone has at least 30 different pairs of sensor/response regulator proteins.(9) Sensor kinases and response regulators are the two principal players of the two-component signaling cascade (Fig. 3) .(9) The sensor kinase (receptor-like) component typically comprises two domains: an amino-terminal input domain, which perceives the signal, and a carboxyl-terminal histidine protein kinase (HPK) domain, which transmits the signal. Sequence comparison of several hundred sensor kinases have indicated that the HPK domains are conserved, whereas the input domains appear to be unrelated modules. The prototypical response regulator consists of a conserved receiver module and a variable output domain that mediates downstream responses. Two-component pathways share the following His-Asp signaling mechanism: signal perception regulates autophosphorylation of a conserved His residue in the sensor kinase, and the phosphoryl group is then transferred to a conserved Asp residue in the receiver domain of the response regulator, thereby modulating the activity of the output domain (Fig. 3). Known output domains include transcription factors or regulators of kinase activities. Numerous configurations of this basic two-step pathway have been identified.(9) In some cases, as in the ethylene receptors, the response regulator is not an independent polypeptide but a carboxyl-terminal domain of the sensor, in which case the receptor is called a hybrid HPK.(9,31) Some hybrid HPKs transmit the signal via a multistep phosphorelay involving a third component known as a histidine-containing phosphotransfer (HPt) domain protein. The HPt protein serves as a phosphohistidine intermediate in the transfer of the phosphoryl group from a receiver domain onto another receiver, resulting in a His-Asp-His-Asp phosphorelay (Fig. 3).(9,31) The ethylene receptor family has sequence similarity to both the sensor kinase and hybrid HPK of the two-component system (Fig. 4). In Arabidopsis, all five ethylene receptors (ETR1, ERS1, ETR2, EIN4, ERS2) have an amino-terminal ethylene-binding domain (described in the next section), and the carboxyl-terminal portion has similarity to HPKs. Three of BioEssays 23.7 621 Review articles Figure 3. Basic features of the two-component signaling system. Signal perception by the input domain of the sensor kinase regulates histidine protein kinase activity. Following autophosphorylation of a conserved histidine residue, the phosphate is transferred to a conserved aspartate residue that is either in the receiver domain of the cognate response regulator (lower solid arrow) or, in the case of a hybrid HPK, in a covalently-attached receiver domain (dashed arrow). In the former situation, the receiver domain directly modulates the output domain, which is the effector for downstream responses. In the His-Asp-His-Asp multistep phosphorelay (dashed arrows), there is a histidine-containing phosphotransfer (HPt) domain protein, which serves as a phosphohistidine intermediate in the transfer of the phosphoryl group from the receiver of the hybrid HPK onto the receiver of a response regulator. the receptors (ETR1, ETR2 and EIN4) also have a covalently attached carboxyl-terminal receiver domain, and are therefore considered to be hybrid HPKs (Fig. 4). ERS1 and ERS2 lack a receiver domain, suggesting that these receptors may have a distinct signaling circuit, or perhaps they signal to a receiver Figure 4. Structure of Arabidopsis ethylene receptor family. The ethylene-binding domain is located in the membrane anchor of the receptors, which comprises either three (solid vertical bars) or four hydrophobic regions. (The fourth region is designated by a shaded bar.) A GAF-related domain of unknown function lies in the presumed cytoplasmic portion, next to the ethylene-binding domain.(63) The table indicates whether the domains are present (Y) or absent (N) for each receptor. All five receptors contain a histidine protein kinase domain, but only ETR1 and ERS1 possess the conserved motifs (N, G1, F and G2)(9) that are considered necessary for histidine kinase activity. ETR1, ETR2 and EIN4 are structurally similar to hybrid histidine protein kinases, since they have a covalently attached carboxyl-terminal receiver domain. 622 BioEssays 23.7 domain of one of the hybrid HPK receptors. To date, the only demonstrated activity (other than ethylene binding) for any of the receptors is in vitro autophosphorylation of ETR1 on the conserved histidine residue.(32) If the ethylene receptors function as prototypical twocomponent signaling proteins, then each of the hybrid HPK receptors should act in a multistep phosphorelay employing an HPt protein followed by a response regulator protein, whereas the non-hybrid receptors would each be paired with a separate response regulator. Several functional HPt homologs,(11,33,34) and over a dozen plant response regulator homologs have been identified,(35) and the signaling roles of these proteins are currently being investigated. Several Arabidopsis HPt homologs can interact with ETR1 in the yeast two-hybrid assay.(36) As presented in the remainder of this article, however, the actual signaling mechanism may be more complex than a twocomponent phosphorelay. For example, ETR1 and ERS1 contain all of the conserved sequence motifs that characterize functional histidine kinases, yet the other three receptors actually lack most or all of these motifs, raising the question of whether the latter three possess histidine kinase activity (Fig. 4). The expected target of two-component signaling, namely an output domain regulated by a receiver domain, is also unknown. Thus, the role of phosphorylation and the extent of similarity to ``classic'' two-component signaling pathways have yet to be established for ethylene signaling. A sequence of unknown function, which has similarity to GAF domains, is situated between the ethylene-binding domain and the HPK domain. GAF domains are found in diverse phototransducing proteins in eukaryotes and prokaryotes, including the plant photoreceptor phytochrome. In some proteins, the GAF domain is involved in binding of cGMP.(37) Since phytochrome is believed to have evolved from prokaryotic two-component histidine kinase photorecep- Review articles tors,(38,39) the ethylene receptor may have arisen from an ancestral prokaryotic photoreceptor upon the addition of an ethylene-binding domain. The genome of the cyanobacteria Synechocystis in fact encodes a sequence that aligns with the input domain of the ethylene receptors, and the protein product is capable of binding ethylene.(40) The encoded protein has no sequence similarity to two-component proteins, however, and its role in Synechocystis is unknown. Ethylene binding Several lines of evidence suggest that ETR1 and its homologs function as receptors for ethylene. Dominant mutations in all five ethylene receptor genes confer pleiotropic ethylene insensitivity,(2,16,18) and examination of etr1-1 mutant plants revealed a reduced ability to bind ethylene.(2) The most compelling argument that these proteins are receptors is that ETR1 and ERS1 reversibly and saturably bind ethylene, as shown using a yeast cell expression system.(15,40,41) The binding occurs with a dissociation constant of 0.04 ml/L gaseous ethylene and a half-life of 12 hours, both of which are consistent with rates observed in ethylene-binding/response assays in plants.(42±44) The ethylene-binding domain of ETR1 lies within the first 128 amino acids ,(15,40) and this domain is the most conserved region among the five Arabidopsis ethylene receptors (44±54% amino acid identity). In the yeast expression system, mutations in this domain can result in the loss of ethylene-binding ability.(15,40,41,45) The binding site appears to consist of three hydrophobic regions, which perhaps serve as a membrane anchor.(40,46) ETR1 indeed localizes to plant membrane fractions,(24) however the identity of the particular membrane is still under investigation. Since ethylene is 14-fold more soluble in lipid than in water, a membrane-localized binding site for ethylene is not unreasonable.(1) A transition metal was predicted to be necessary for ethylene binding,(48) and consistent with this hypothesis, ETR1 was shown to bind ethylene only in the presence of copper ions.(40,45) Interestingly, a gene involved in the proper functioning of the ethylene receptors, RAN1, codes for a copper transporting P-type ATPase.(49,50) ran1 mutants develop a constitutive ethylene-response phenotype similar to that of multiple receptor loss-of-function mutants (which are discussed later). The RAN1 protein is located in intracellular membrane compartments and is believed to deliver copper ions from storage sources inside the cells. ran1 loss-offunction mutants display a rosette lethal phenotype, which is thought to be caused either by general effects due to the reduced copper availability for other copper-utilizing enzymes, or by disruption of ethylene receptor function.(49,50) Notably, plants containing loss-of-function mutations in four of the five ethylene receptor genes (all but ERS1) also often died before flowering.(51) Inhibition of receptor signaling by ethylene An interesting feature of the ethylene receptors is that they are negative regulators of the ethylene-response pathway, i.e. they repress ethylene responses in the absence of the ethylene signal (Fig. 5). This is counterintuitive to the notion of receptors being activated by their signals to turn on responses. The negative regulation by the ethylene receptors was revealed through the analysis of severe loss-of-function or null mutants, which were isolated for four of the Arabidopsis ethylene receptor genes.(51) (Loss-of-function mutants for ETR1, ETR2 and EIN4 were identified among revertants of dominant ethylene-insensitive mutants for these genes, and a loss-of-function ERS2 allele was obtained by a T-DNA insertion. An ERS1 loss-of-function mutant has not yet been described.) Single ethylene receptor mutants showed essentially wild-type phenotypes, thus indicating that the genes have redundancy. In contrast, when the receptor mutants were crossed together, the resulting homozygous triple and quadruple loss-of-function mutants displayed constitutive ethylene responses. Since the absence of receptors resulted in constitutive responses, the wild-type receptors must be active repressors (i.e. negative regulators) of ethylene responses. This suggests a model in which in the receptors repress ethylene responses when ethylene is unbound (Fig. 5). Consequently, when ethylene is bound, the receptors are inactivated leading to ethylene responses.(51,52) These results provide a probable explanation for why only dominant ethylene receptor mutants have been isolated in Figure 5. Model for ethylene receptor action. The receptors are negative regulators of the ethylene signaling pathway, since multiple receptor loss-of-function mutants develop a constitutive ethylene-response phenotype. When ethylene is not bound (A), the receptors interact and activate CTR1, which results in the inhibition of ethylene responses. When ethylene is bound (B), the receptors are inactive with respect to activating the downstream CTR1 protein. The ethylene insensitivity of the dominant receptor mutants is caused by a receptor protein that is constitutively active due, in some cases, to the inability to bind ethylene. BioEssays 23.7 623 Review articles mutant screens. Any single loss-of-function receptor mutants would be wild-type in appearance, whereas dominant receptor mutants could be isolated by virtue of their ethyleneinsensitive phenotype. Since the dominant mutant phenotype is opposite that of the triple and quadruple loss-of-function mutants, it follows that the dominant receptor mutants are caused by gain-of function mutations. (Note that the gain of function is with respect to signaling activity, not with respect to ethylene binding.) A basis for the dominance of the ethylene-insensitive receptor mutations Based on the conclusion that ethylene receptor signaling is active in the absence of ethylene binding and inactive when ethylene is bound, we can attempt to derive a model for how the dominant mutations confer insensitivity to ethylene. Each of the dominant ethylene receptor mutations results in an amino acid substitution in the ethylene-binding domain, and several of these mutations correlate with a dramatic reduction in ethylene binding.(45) Conceivably, the inability to bind ethylene locks the signaling domain (cytoplasmic portion) into a perpetually active signaling state, such that responses are constitutively repressed despite the presence of redundant wild-type family members. Thus, the mutation would be dominant rather than recessive. This explanation, however, is unlikely to be the entire basis for the ethylene-insensitive phenotype. First, the repression activity would have to be very strong, because several of the dominant mutants are completely insensitive to ethylene, even when treated with extremely high doses of ethylene. Second, the fact that we see constitutive ethylene responses in the quadruple or triple lossof-function mutants (which still carry one or two wild-type receptor genes, respectively) suggests that having one or two receptors is insufficient to repress responses. In other words, the dominant ers1-1 mutant (which carries four redundant wild-type receptor genes) strongly inhibits the ethyleneresponse pathway,(16) yet the single wild-type ERS1 gene cannot prevent the constitutive ethylene response in the quadruple receptor mutant.(51) Therefore, it has been proposed that the mutant gain-of-function receptors are somehow hyperactive.(46,51,52) We speculate that hyperactive signaling by the dominant mutant receptors might be achieved via higher order complexes of ethylene receptors, such that the locked signaling conformation of a mutant receptor dictates the conformation of the associated receptors. The complexes might comprise either multiple receptor homodimers, heterodimers or higher multimers. There is no evidence yet for or against heterodimer formation, although ETR1 antibodies detect the ETR1 dimer at a single specific molecular weight in Arabidopsis membrane extracts.(47) Evidence from yeast two-hybrid experiments suggest that the GAF domains of different ethylene receptors (e.g., ETR2 and ETR1) can associate with each other 624 BioEssays 23.7 (C.K. Wen and C. Chang, unpublished). The capacity for dimerization is also suggested by the three dimensional structure of the ETR1 receiver domain.(53) Bacterial histidine kinases have been shown to form heterodimers and catalyze transphosphorylation.(54,55) Conceivably, a dominant mutant ethylene receptor might catalyze transphosphorylation of wildtype ethylene receptors, which would otherwise be inactive. Why are there multiple ethylene receptors? The genetic data in Arabidopsis indicate that the receptor genes have functional redundancy with respect to the seedling triple response. Single null mutants appear to be wild type, while the dominant receptor mutations result in a similar phenotype independent of which receptor gene is mutated. The expression patterns of the receptor genes also overlap widely.(17) Families of ethylene receptor genes are found not only in Arabidopsis, but in other plants as well. So, what might be the advantage for having multiple receptors? The fact that ethylene sensitivity varies between tissues and for different responses suggests a rationale for having multiple receptors.(44) As suggested by Hua and Meyerowitz,(51) the receptors may differ in their affinity for ethylene, enabling the plant to respond to a broad spectrum of ethylene concentrations. In addition, the expression patterns of ethylene receptor genes in tomato implicate tissue-specific roles for the different genes.(56,57) For example, an ERS1 homolog is highly upregulated during tomato fruit ripening.(56) Expression of three of the Arabidopsis ethylene receptor genes are also induced by ethylene.(17) The upregulation of receptors in response to ethylene might provide a mechanism for adaptation to ethylene; since the half-life of ethylene binding is very long, an increase in the number of unbound receptors may enable the plant to react to changes in ethylene concentration. Linking the ethylene receptors to CTR1, a MAPKKK The next known component downstream of the ethylene receptors is CTR1, which is thought to function as a MAPKKK. In animals, MAPKKKs are typically regulated by tyrosinekinase receptors or by G-protein-coupled receptors.(58) There are only two known examples of pathways that combine a MAPK pathway with a two-component system. One is the S. cerevisiae osmosensing pathway(21,59) and the other is the S. pombe stress-response pathway.(22,23) In the osmosensing pathway, the hybrid histidine kinase Sln1 transfers a phosphoryl group from the Asp residue in the Sln1 receiver domain to a His residue in a separate HPt protein called Ypd1, and then to an Asp in the response regulator protein Ssk1. Ssk1 interacts with and regulates two downstream acting MAPKKKs, Ssk2 and Ssk22.(21,59) The ethylene signaling pathway may differ from this yeast pathway, since a direct interaction has been observed Review articles between CTR1 and both ETR1 and ERS1.(60) The CTR1 amino-terminal domain, which is the presumed regulatory domain, can associate with the histidine kinase domain of ETR1 and ERS1 as well as the ETR1 receiver domain, as shown by the yeast two-hybrid assay and by in vitro copurification techniques. It is not yet known whether these components alone are sufficient to transfer the signal from the receptors to CTR1. In the yeast osmosensing pathway, the unphosphorylated form of the response regulator SSK1 appears to directly activate the MAPKKK Ssk2.(21,59) For the ethylene-response pathway, there may be a similar mechanism involving the ETR1 receiver domain and CTR1. Recently, the three-dimensional structure of the ETR1 receiver domain was determined.(53) The structure highly resembles the 3D structure of previously investigated bacterial receivers such as Escherichia coli Che Y, even though the sequence identity between ETR1 and CheY is only 17.6%. Interestingly, receiver domains (including that of ETR1) show striking structural similarity to Ras proteins,(53) which are involved in activating Raf kinases (to which CTR1 is similar). Since Ras and Raf are known to interact, the mechanistic link between ETR1 and CTR1 could be similar to the signaling between Ras and Raf. CTR1 might be activated by dimerization facilitated by ETR1. As mentioned earlier, evidence suggests that ETR1 forms homodimers through disulfide linkages in the ethylenebinding domain.(47) In addition, the ETR1 receiver domain forms homodimers both in solution and in the crystal, as observed for several other bacterial receivers. For some receivers, dimerization is independent of the phosphorylation state, whereas in others, dimerization only occurs in the unphosphorylated state. The interaction interface in the ETR1 receiver dimer shows high structural similarity to the interface of phosphorylation-dependent dimerizing receivers. Consequently, Mueller-Dieckmann et al.(53) proposed that dimerization of the ETR1 receiver domain occurs in the unphosphorylated state, and that phosphorylation would cause monomerization. Adding CTR1 to the picture, we speculate that ethylene binding causes autophosphorylation of ETR1, resulting in the inactivation of the CTR1 kinase (which is a negative regulator of ethylene responses) perhaps through the loss of dimerization. That is, if CTR1 is associated with ETR1, then CTR1 might be dimerized and thus activated in the absence of ethylene, and the monomerization of the ETR1 receiver domain upon ethylene-induced phosphorylation might cause loss of CTR1 dimerization, thereby inactivating CTR1. An association between CTR1 and the ethylene receptors does not rule out the participation of other proteins acting in a multimeric interaction complex as shown in bacterial chemotaxis.(61) In addition, it is worth noting that ctr1 null mutants are still capable of responding to ethylene, indicating the existence of an alternative pathway for ethylene responses.(62) Thus, a direct regulation of CTR1 does not preclude a ``traditional'' two-component signaling pathway leading from the receptors. Conclusions Great progress has been made in understanding ethylene perception and signaling through the use of genetic approaches in Arabidopsis. However, much remains to be uncovered concerning the biochemical functions of and interactions among the different ethylene receptors. It is still unclear what role phosphorylation plays in the response pathway and how the presence and absence of ethylene is relayed to the cell by the different receptors. For example, does binding of ethylene modulate histidine kinase activity and, if so, in what manner? Does phosphorylation-dependent receptor dimerization occur? We know that the receptors repress ethylene responses, however, the mechanism for this repression is unknown. Perhaps repression does not involve a biochemical activity, and inactivation of the repression occurs through histidine kinase activity elicited by ethylene binding. It is also important to determine the distinct role provided by each member of the ethylene receptor family. For this, it will be essential to have a null mutant of ERS1Ðperhaps ERS1 is a positive regulator of ethylene responsesÐand to describe the phenotype of plants that completely lack all five receptor genes. The subcellular location of the receptors has yet to be determined. Differences in tissue localization may help to reveal different functions among the five receptors. Another question that should be addressed is the process of adaptation to ethylene. This may involve recycling of receptors, protein degradation and/or synthesis of new receptors. Finally, new components, such as regulators of receptor function, and substrates for the receptors have yet to be revealed. Recent advances in reverse genetics and Arabidopsis genomics should speed the discovery of such components. Acknowledgments We thank our colleagues Stephen Mount, Richard Stewart and Zhongchi Liu for discussion and comments on the manuscript. References 1. Abeles FB, Morgan PW, Salveit ME. ed; Ethylene in Plant Biology. San Diego. Academic Press. 1992. 2. Bleecker AB, Estelle MA, Somerville C, Kende H. Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 1988;241:1086±1089. 3. GuzmaÂn P, Ecker JR. Exploiting the triple response of Arabidopsis to identify ethylene-related mutants. Plant Cell 1990;2:513±523. 4. 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