The ethylene receptors: Complex perception for a simple gas

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Plant Science 175 (2008) 8–17 www.elsevier.com/locate/plantsci

Review

The ethylene receptors: Complex perception for a simple gas Brad M. Binder * Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706, United States Received 17 October 2007; received in revised form 5 December 2007; accepted 7 December 2007 Available online 15 December 2007

Abstract Ethylene is a hormone that regulates a number of physiological and developmental events in plants. This simple olefin is perceived by a family of receptors that have homology to bacterial two-component receptors. Although the ethylene receptors have been the focus of much study over the past several decades, many questions remain concerning their structure, function, and regulation. This review provides an overview on what is known about the ethylene receptors, summarizes recent research on the receptors, and presents models for receptor function and output. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethylene; Receptors; Transition metals; Two-component; Signal transduction; Protein kinase

Contents 1. 2. 3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . General overview of the receptors . . . . . . . . . . The ethylene binding domain . . . . . . . . . . . . . Receptor output . . . . . . . . . . . . . . . . . . . . . . . 4.1. Receptor kinase activity . . . . . . . . . . . . 4.2. Physical interactions with CTR1 . . . . . . 4.3. Other interactions? . . . . . . . . . . . . . . . . Non-redundant receptor function . . . . . . . . . . . Receptor–receptor interactions/receptor clusters . Receptor expression and degradation . . . . . . . . Conclusions and future directions . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Ethylene is a simple, unsaturated hydrocarbon. Despite its chemical simplicity, ethylene affects many diverse processes throughout the lifetime of a plant including seed germination, growth, formation of the apical hook, organ senescence, fruit

Abbreviations: AHP, Arabidopsis histidine phosphotransfer proteins; CTR, constitutive triple response; EIN, ethylene insensitive; ERS, ethylene response sensor; ETR, ethylene response; RAN, responsive to agonist; RTE, reversion to ethylene-sensitivity. * Tel.: +1 608 262 1543; fax: +1 608 262 4743. E-mail address: [email protected]. 0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2007.12.001

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ripening, abscission, gravitropism, and responses to various stresses [1,2]. Neljubov [3] is credited with the discovery that ethylene is biologically active. He showed that ethylene was the active compound in illuminating gas that caused horizontal growth of etiolated pea seedlings. While research over the decades following this discovery expanded upon this to document a large number of responses affected by ethylene, it was not until the late 1970s that ethylene binding sites were found in plants and subsequently biochemically characterized [4–9]. However, purification and thorough characterization of the receptors isolated from plants was hampered by a number of factors including membrane localization, low expression levels, and the presence of multiple isoforms. The use of Arabidopsis

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as a model plant system to study ethylene signalling [10,11] and the cloning of the ETR1 ethylene receptor from Arabidopsis [12] opened up a new avenue of research that has yielded a large amount of information about ethylene signalling including the receptors. This review provides an overview of what is known about the receptors and focuses on our current understanding of the ethylene receptors from Arabidopsis. 2. General overview of the receptors Responses to ethylene are mediated by a family of receptors. The number of receptor isoforms varies from species to species [13–29]. In Arabidopsis thaliana where most research has been conducted, there are five receptor isoforms all of which can bind ethylene [30–32]. However tomato (Solanum lycopersicon or Lycopersicon esculentum) is emerging as another important system to study ethylene receptor function. In tomato there are six receptor isoforms [33–36] five of which have been tested for ethylene binding and found to bind ethylene with high affinity [32]. A survey for ethylene-binding activity in members from different kingdoms indicates that high-affinity ethylene binding sites are found in land plants, the alga Chara spp., and a group of cyanobacteria but are essentially absent from individuals in other kingdoms [37]. Bioinformatic analysis shows that ethylene binding like sequences are highly represented in plant and cyanobacterial species. This together with the distribution of ethylene binding is consistent with the hypothesis that the ethylene receptors have a plastid origin [37–39]. Despite the varying number of receptor isoforms in different species, the different isoforms share many structural features in common (Fig. 1). The receptors form homodimers that are stabilized at their N-termini by two disulfide bonds [31,40,41]. All are predicted to contain three N-terminal transmembrane domains, a GAF domain of unknown function, and a kinase domain. A subset of the receptors also contain a receiver domain at their C-termini. The ethylene receptors in plants can be divided into two subfamilies (Fig. 1). In Arabidopsis, subfamily I consists of AtETR1 and AtERS1, that contain all amino acid residues needed for His kinase activity [12,42] and show His kinase activity in vitro [43,44]. Subfamily II includes AtETR2, AtEIN4, and AtERS2, that contain degenerate His kinase domains [45,46] and have Ser/Thr kinase activity in vitro. AtERS1 is bifunctional displaying both His and Ser/Thr kinase activities in vitro [44]. Subfamily II receptors contain an additional putative transmembrane domain that may function as a signal sequence affecting localization of the receptors. A third subfamily is comprised exclusively of putative ethylene binding proteins from cyanobacteria [37]. Ethylene binding sites in bean (Phaseolus vulgaris) [6,7] and specific receptor isoforms from Arabidopsis [47,48] and melon (Cucumis melo) [49] have been localized to the membranes of the endoplasmic reticulum. Studies on CmERS1 from melon confirms that it has three transmembrane domains with the N-terminus towards the lumen of the endoplasmic reticulum [49]. However, an isoform from tobacco (Nicotiana tabacum) has been localized to the plasma membranes [50].

Fig. 1. Ethylene receptor structure and families in Arabidopsis (At) and tomato (Le). Based on sequence comparisons of the ethylene binding domains, the different ethylene receptor isoforms in plants separate into two subfamilies. All the receptors share similar structures containing an ethylene binding domain, GAF domain and kinase domain. A subset also contain a receiver domain. Members of subfamily II all contain an extra putative N-terminal signal sequence.

These differences could reflect differences in species, receptor isoform examined, or methodological discrepancies. At this point it is also difficult to preclude localization of minor amounts of receptors to the membranes of other organelles such as the Golgi [47]. Specific, gain-of-function mutations in the binding domain in any of the Arabidopsis receptor isoforms confer dominant ethylene insensitivity in the plant indicating all contribute to signalling [10,12,42,45,46,51]. Loss of multiple receptor isoforms results in constitutive ethylene responses [52]. CTR1 is immediately down-stream of the receptors and genetically acts as a negative regulator of the pathway [53,54]. From these studies, an inverse agonist model for ethylene signal transduction has been developed. In this model, in air the receptors stimulate CTR1, which inhibits down-stream components in the pathway. This means that the receptors (via CTR1) are negatively regulating the pathway. Interestingly, ethylene inhibits the receptors. This leads to decreased activity of CTR1, which releases down-stream components from inhibition by CTR1. Put another way, ethylene inhibits an inhibitory step in the signalling pathway leading to ethylene responses. Thus, in this model, gain-of-function mutations in the receptors lead to constitutive receptor output causing CTR1 to constitutively inhibit the pathway. This results in ethylene insensitivity. Conversely, loss of receptors leads to a decrease in signalling from the receptors (much like addition of ethylene) causing reduced output from CTR1. This releases components down-stream of CTR1 from inhibition and causes constitutive ethylene responses.

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3. The ethylene binding domain The first step in ethylene signalling occurs when ethylene binds to the receptors. Studies on exogenously expressed, truncated versions of the receptors from Arabidopsis [30,32,55] and tomato [32] show that the ethylene binding domain is contained within the N-terminal transmembrane domains. Cu(I) ions are required for high-affinity ethylene binding in AtETR1 receptors exogenously expressed in yeast [55] supporting earlier speculations about the requirement for a transition metal cofactor for ethylene binding [56,57]. Genetic studies find that the RAN1 copper transporter acts upstream of the receptors [58,59] consistent with a model where copper ions are delivered to and required by the ethylene receptors. There is one copper ion per receptor dimer suggesting that dimers form the functional unit for the receptor [55]. Interestingly, the disulfide bonds at the N-termini are not required for ethylene signalling [60]. However, dimerization could be occurring through interactions between other domains including the GAF domain [60], receiver domain [61] or even non-specific interactions between the hydrophobic transmembrane domains. It is thought that ethylene binding causes a change in the coordination chemistry of the Cu(I) cofactor. This, in turn, results in a change in the binding site that is transmitted through the receptor dimer to down-stream signalling elements. Copper is a member of the Group 11 transition metals. Interestingly, of numerous other transition metals tested, only the other two Group 11 metals (silver and gold) can substitute as a cofactor for ethylene binding to exogenously expressed AtETR1 [55,62]. Since silver ions block the action of ethylene in plants [57], a model has developed where silver can replace copper in the receptor binding pocket yet prevents changes in the receptor required for transmission of the signal through the receptors. Consistent with this idea, Ag(I) ions are approximately 70% larger than Cu(I) ions [63]. However, gold ions do not block the action of ethylene in plants [62]. While a simplistic prediction based upon atomic mass is that Au(I) has a larger ionic radius than Ag(I), Au(I) is actually smaller than Ag(I) [63–65]. Also, unlike copper and silver ions where only the Cu(I) and Ag(I) valence states can complex with olefins, two valence states of gold form complexes with olefins so that Au(III) also binds to ethylene [66,67]. Au(III) is much smaller than Ag(I) being only slightly larger than Cu(I). Thus, it is possible that gold ions do not block ethylene signalling simply because they are smaller than silver ions. Analyses of mutations in the transmembrane domain of AtETR1 have revealed that certain residues are crucial for ethylene binding [30,51,55]. Interestingly, the etr1-1 mutant containing a Cys65Tyr mutation is incapable of binding copper and ethylene leading to the idea that this residue forms part of the ethylene/copper binding pocket [55]. These earlier observations have been extended by a recent study on the ethylene binding domain of AtETR1 [37]. These workers made mutations in amino acids of the AtETR1 ethylene binding domain that are conserved between the Arabidopsis receptors and Synechocystis slr1212 and examined ethylene-binding activity in exogenously expressed protein as well as the ability

of mutated receptors to rescue growth and signalling defects in receptor null plants. Mutations in certain amino acid residues lead to altered receptor function. One class of mutations in specific amino acid residues in the middle regions of Helices 1 and 2 eliminate ethylene binding and confer constitutive receptor signalling. These residues align along a single face of their respective helix suggesting they come together to form the copper/ethylene binding pocket (red in Fig. 2). A second class consists of mutations in amino acids residues that cluster near the cytoplasmic ends of Helices 1 and 3 and yield a receptor with normal ethylene-binding activity yet still confer constitutive signalling. Thus, these residues may have a role in turning off the signal transmitter domain of the receptor when ethylene binds (green in Fig. 2). The etr1-2 mutation has an Ala102Thr substitution and belongs to this class of mutants [51]. Two mutations at the cytoplasmic end of helix 3 yield a receptor that is loss-of-function being incapable of signalling suggesting they are required for the ‘‘transmitter on’’ conformational state (blue in Fig. 2). These results have led to an updated model of receptor structure-function with respect to ethylene binding that posits there is an intermediate state to the receptor when ethylene binds (State 2 in Fig. 2). This intermediate state has ethylene bound but is still signalling and likely represents an unstable conformation of the receptor. While ethylene is bound, this conformational State 2 is in equilibrium with the more stable conformational State 3 where ethylene is bound and signalling is inhibited. The existence of such an intermediate conformation could explain why receptor null mutations cause a more severe constitutive response phenotype than wild-type plants saturated by ethylene [52,68,69]. In this model, a small percentage of receptors in the presence of saturating concentrations of ethylene are in conformational State 2 and still signalling whereas receptor null plants lack receptors in this signalling state. While these studies give us much more information about regions of the ethylene binding domain that are important for stimulus–response coupling, the specific nature of the conformational changes associated with signalling is unknown. Possible motions of the a-helices include translation, pistoning, pivoting, and rotation perpendicular to the membrane [70]. Since the receptors form dimers, conformational changes associated with ethylene binding may involve intramolecular realignments between helices within a monomer or intermolecular realignments between monomers in the dimer pair. 4. Receptor output 4.1. Receptor kinase activity Ethylene receptors have homology to bacteria twocomponent receptors [12] that function via histidine autophosphorylation followed by phosphotransfer through a conserved aspartate on a receiver domain or separate response regulator [71]. The observation that the Arabidopsis subfamily I receptors play a more prominent role in signalling coupled with the fact that these receptors contain functional His kinase

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Fig. 2. Receptor signal state model. The ethylene binding domain of ETR1 is depicted as a homodimer with each monomer containing three transmembrane helices. Areas that affect ethylene signalling have been defined by mutational analyses and are shown. These include regions important for copper/ethylene binding, turning off the receptor, and maintaining the receptor in the signalling state. In air, the receptor is signalling (State 1). When ethylene binds there is an intermediate conformation where the receptor is still transmitting signal (State 2). This is predicted to be an unstable conformation. While ethylene is bound it is in equilibrium with State 3 where ethylene is bound and signal transmission is turned off. Adapted from Wang et al. [37].

activity [43,44,72] initially led to the idea that His kinase activity is important for ethylene signal transduction. While the kinase domain of AtETR1 is required for proper receptor function [60,73], His kinase activity per se is not [60,69,72,74]. However, His kinase activity and the receiver domain may affect ethylene signalling behaviour in more subtle ways. For instance, AtETR1 His kinase activity appears to modulate growth [73,75] and ethylene-sensitivity [73]. The receiver domain may also have a role in modulating sensitivity to ethylene [73]. Additionally, Binder et al. found that AtETR1 His kinase activity and phosphotransfer through the receiver domain are involved in recovery of growth after removal of ethylene [74]. Since His kinase activity is not required for the primary ethylene response but may play subtle roles in signalling, it is possible that receptor His kinase activity modulates output from the ethylene signalling pathway or it could be involved in modulating other two-component signalling pathways. Some evidence for signalling cross-talk between ethylene receptors and other two-component pathways in plants has been published but the function of this is still unclear [75–78]. Ser/Thr kinase activity has been observed in certain receptor isoforms from Arabidopsis [44] and tobacco [50,79]. Therefore, it is possible that receptor Ser/Thr kinase activity influences receptor signalling, perhaps by modulating CTR1 activity. However, attempts to test this by eliminating kinase

Ser/Thr kinase activity in the receptors have so far been unsuccessful leaving it unknown whether or not Ser/Thr kinase activity has a role in ethylene receptor output. In addition to the open questions above, it is also unknown whether or not ethylene binding to the receptor actually alters receptor kinase activity. While the action of ethylene has been genetically defined as a negative regulator, at the biochemical level ethylene could be leading to either an increase or decrease in kinase activity to affect CTR1. Thus, there is more work required to resolve the role(s) that receptor protein kinase activity has in ethylene receptor function. Potential roles for the kinase activity include signal output, signalling cross-talk, or regulating receptor degradation. 4.2. Physical interactions with CTR1 While ETR1 His kinase activity appears to play a small role in ethylene signalling, the His kinase domain is required for proper receptor function [60,73]. This raises the question, what aspects of this domain are important for ethylene receptor function? One possibility is that the kinase domains physically interact with down-stream signalling components to control the pathway. A number of studies using several methods have now documented a physical interaction between the receptors and CTR1 [53,80–82]. Yeast two-hybrid studies found that AtETR1 and AtERS1 have a stronger interaction with CTR1 than

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AtETR2 [80,81]. Interestingly, interactions of the AtETR1 and AtERS1 receptors with CTR1 require the His kinase domain [81,82] consistent with the observation that this domain is required for receptor function. His kinase activity is not required for the interaction between AtETR1 and CTR1 [82]. Like the receptors, CTR1 is associated with the ER membranes and perhaps additional membranes such as the Golgi [82]. Double and triple receptor loss-of-function mutants that lead to constitutive ethylene responses have reduced levels of CTR1 associated with the membranes showing that this association is receptor-dependent [82]. The ctr1-8 constitutive response mutant has a missense mutation in the N-terminus that affects its interaction with AtETR1 [53] and results in a protein that fails to associate with membranes [82]. Together these studies suggest that the kinase domains of the receptors associate with the N-terminus of CTR1. While it would be tempting to invoke dissociation of CTR1 from membranes as the mechanism for ethylene signalling, this is unlikely since ethylene treatment actually results in an increase in CTR1 associated with the membranes [82]. 4.3. Other interactions? Though interactions between the receptors and CTR1 have received the most attention, it is likely that the receptors interact with other proteins including other receptor isoforms. However, very little has been published to date in this area. As mentioned above, phosphotransfer from the ethylene receptors to other two-component systems might modulate ethylene responses. One study [77] using yeast two-hybrid interactions showed that ETR1, but not ERS1, interacts with several histidine phosphotransfer proteins (AHP1,2,3). The role of these interactions is unclear. There is also physiological evidence showing that authentic histidine kinase 5 from Arabidopsis modulates ethylene responses in the roots but not hypocotyls [78]. However, it is an open question whether this modulation occurs at or down-stream of the receptors. There is now genetic evidence that a newly discovered class of proteins is a positive regulator of the ETR1 receptor [83,84]. In Arabidopsis, RTE1 appears to function primarily through ETR1 and restores ethylene-sensitivity to the etr1-2 mutant [84]. A similar gene (Green-ripe) has been found in tomato and is involved in responses to ethylene including ethylenedependent fruit ripening [83]. rte1 null mutants and rte1 etr1 double null mutants have enhanced responses to ethylene indicating that both act in the same pathway [84]. RTE1 has been localized to the membranes of the Golgi [85,86] and endoplasmic reticulum [85] and co-localizes with ETR1 in these membranes thus supporting a model where RTE1 modifies ETR1 function [85]. It is still not known if these proteins interact directly with receptors, but it is interesting to note that the His kinase and receiver domains of AtETR1 are not needed for RTE1 functioning while the N-terminus of RTE1 is required [86]. It is also of interest that RTE1 does not restore sensitivity to the etr1-1 ethylene-insensitive mutant [84]. This is intriguing since, as mentioned above, the etr1-2 mutant protein still binds ethylene but is incapable of turning off upon

binding of ethylene suggesting RTE1 may function in this conversion from the ‘‘signalling on’’ to ‘‘signalling off’’ state of the receptor. It will be interesting to see if RTE1 directly interacts with ETR1 or any of the other receptor isoforms and whether amino acid residues needed for the receptor transmitter off state affect RTE1 function or interactions with ETR1. 5. Non-redundant receptor function All the receptors contribute to signalling and there is functional overlap between the different isoforms. However, they are not entirely redundant in function. Perhaps the clearest example of this is the observation that AtETR1 has a nonoverlapping role from the other receptor isoforms in ethylenestimulated nutational bending of Arabidopsis hypocotyls [87]. However, other examples exist. For instance, various combinations of triple loss-of-function receptor mutants display a constitutive ethylene response phenotype [32,52,68] yet these plants have wild-type levels of total receptor transcript and ethylene binding [32]. This shows that even though the mutant plants compensate for the loss of specific receptor isoforms by increasing the expression of the other isoforms, this does not functionally rescue the plants. Another example of nonredundant function is the non-equivalent roles that AtETR1 and AtEIN4 have in the suppression of the rhd1 root hair phenotype by ethylene [88]. More evidence for non-redundancy is found in the observation that particular receptor isoforms have a more prominent role in ethylene signalling. In Arabidopsis etr1;ers1 double loss-offunction mutants have a more severe constitutive ethylene signalling phenotype than other double loss-of-function mutants [68]. Earlier studies were done using the ers1-2 mutant that has been found to have residual expression of ERS1. More recent experiments using the complete loss-of-function ers1-3 mutant have largely replicated the earlier results [60,89]. Thus, the subfamily I receptors appear to have a larger role in ethylene signalling in Arabidopsis. Interestingly, Ag(I) appears to mainly target subfamily I receptors to prevent responses to ethylene [60,80]. Based on these observations coupled with interaction studies between the receptors and CTR1, a model has been proposed [89] where the subfamily I receptors physically interact with and activate CTR1 more strongly than subfamily II receptors. Consistent with this model, etr1-6;ers1-1 mutants have a stronger constitutive ethylene response phenotype compared to etr2-3;ein4-4;ers2-3 mutants [68]. Although all the components for ethylene signal transduction are shared between plant species, there are differences to note even at the level of the receptors. In addition to differences in the number of receptor isoforms between species that was mentioned above, other differences are becoming evident. For instance, regarding the importance of specific isoforms a different pattern has emerged in tomato where LeETR4 or LeETR6 antisense plants cause early ripening while other receptor antisense plants do not ripen early [90,91]. Both LeETR4 and LeETR6 are subfamily II receptors showing that in tomato, the subfamily I receptors do not have a predominant role in ethylene signalling leading to fruit ripening. However,

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subfamily I receptors may play other roles in tomato [92]. This highlights the need to examine a variety of species and developmental processes to gain a full understanding of ethylene receptor function. None-the-less, these studies do point out a non-redundant role for the receptors. Together, these observations support a model where the receptors have overlapping but distinct roles in ethylene signalling. The basis for these unique functions is unclear but could involve unique functional outputs, protein interactions, distribution within the plant, or some combination of these factors. 6. Receptor–receptor interactions/receptor clusters Plants respond to ethylene over a wide concentration range. In Arabidopsis responses to ethylene between 0.2 nL L 1 and 100 mL L 1 have been reported [93,94]. Based on the published Kd for the AtETR1 receptor [30], this means that a response can occur when approximately 0.1% of the receptors bind ethylene. Since there are multiple receptor isoforms in many plants, this ability to respond to such a large concentration range could simply reflect differing Kd’s for each isoform. However, this is unlikely since the different receptor isoforms from both Arabidopsis and tomato appear to have similar affinities for ethylene [32]. This capacity to respond to small changes in ethylene concentration is similar to the behaviour seen in the evolutionarily related bacterial two-component receptors [95]. Models for bacterial chemotaxis propose that amplification results from receptor dimers forming clusters where the occupancy state of one dimer can shift the signalling states of surrounding receptor dimers within a cluster through physical interaction [96–98]. Thus receptor occupancy at low ethylene concentrations could be amplified, leading to a large change in total receptor output allowing plants to sense small changes in receptor occupancy. Receptor clustering has been invoked to explain other observations. For instance, the rate of growth recovery after the removal of ethylene appears to occur faster than dissociation of

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ethylene from the receptors and receptor turnover [74]. Here, small numbers of receptors without ethylene bound are predicted to convert neighbouring ethylene-bound receptors to the signalling state leading to faster recovery of growth. While amplification of signal can occur at any point in the ethylene signalling pathway, the observation that His kinase activity and the receiver domains are required for normal recovery rate after the removal of ethylene suggests that the receptors are playing a direct role in amplification of signal [74]. A receptor-clustering model has also been invoked to explain the observation that a truncated etr1-1 confers high levels of ethylene insensitivity [72]. In a clustering model the mutant, truncated receptor is converting neighbouring receptors to the signalling state even in the presence of ethylene. Receptor clustering may also underlie the observation that specific missense mutations in any of the receptors from Arabidopsis, including isoforms such as EIN4 that are expressed at very low levels, results in an ethylene-insensitive plant [45]. In this model (Fig. 3), ETR1 and ERS1 in Arabidopsis are the primary signalling receptors while the subfamily II receptors act cooperatively to enhance signalling of the subfamily I receptors [99]. Thus, a missense mutation in a poorly expressed receptor like EIN4 would still cause a dominant insensitive plant because it is keeping ETR1 and ERS1 in the signalling state in the presence of ethylene. Since subfamily I double mutants still respond at reduced levels to ethylene [89] this model also hypothesizes that the subfamily II receptors can act independently, albeit at reduced levels, of the subfamily I receptors. As discussed above, the stronger physical interaction between CTR1 and subfamily I receptors versus between CTR1 and subfamily II receptors provides a mechanism to explain the larger importance of subfamily I receptors in Arabidopsis [89]. While there is no direct evidence for ethylene receptor clustering, it is possible that physical interactions between receptor dimers could occur via the GAF or receiver domains [60,61]. The intermediate receptor State 2 mentioned above (Fig. 2) provides a mechanism by which a small number of unbound receptors in a large cluster could alter the signalling

Fig. 3. Receptor signalling model in Arabidopsis. In this model, the subfamily II receptors predominantly signal through the subfamily I receptors. However, subfamily II receptors also interact with and stimulate CTR1, but at reduced levels compared to subfamily I receptors. Receptor domains are depicted as in Fig. 1. Adapted from Binder and Bleecker [99].

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states of surrounding ethylene-bound receptors or conversely, how a small number of bound receptors could alter the signalling state of surrounding unbound receptors [37]. 7. Receptor expression and degradation The inverse agonist model for ethylene signal transduction predicts that ethylene response magnitude and sensitivity increases when the number of receptors decreases. Support from this has been seen experimentally [52,68,80,89,91,100]. Conversely, increasing expression of receptors leads to a decrease in sensitivity [91]. A number of studies have also documented changes in receptor transcript levels during development or in response to stimuli [13–15,17– 29,33,34,41,74,91,101–105]. While these studies give a general picture about the receptor transcript levels and ethylenesensitivity, very few studies examined the receptors at the protein level under these conditions [103]. This is likely to be important since transcript levels do not necessarily reflect protein levels and as mentioned above, the receptors have both redundant and non-redundant functions. Two recent papers have examined post-transcriptional control of receptors in more detail. In work from the Schaller lab at Dartmouth College [48], it was found that low levels of ethylene increase the levels of both the transcript and protein of ETR2 in Arabidopsis. In contrast, at higher ethylene concentrations AtETR2 transcript levels increase but protein levels decrease. A similar story for control of the receptors is also emerging from work on tomato. In a paper from the Klee lab at the University of Florida [90], the role of ethylene receptor turnover in the ripening of tomato fruit was reported. Three of the six receptors (LeETR3, LeETR4, LeETR6) show an increase in transcript levels as ripening progresses. These genes were also found to be induced by high levels of ethylene. However, when the protein levels of each of these receptors was examined, a different picture emerged. LeETR3, LeETR4, and LeETR6 protein levels are high in immature fruits but decline during ripening. Application of ethylene also causes a decrease in the protein levels of these three receptors. Both studies found that ethylene perception is required for this increased breakdown of ethylene receptors and inhibitors of 26S proteasome function block this degradation [48,90]. While these results seem to contradict earlier work showing a correlation between receptor transcript levels and ethylene-binding activity in Arabidopsis [32], this earlier study was done in air where presumably receptor degradation had not been stimulated. Also, ethylene-binding activity is a reflection of total receptor levels, rather than the level of particular receptor isoforms. This increased receptor turnover is predicted to control ethylene-sensitivity. As discussed above, the receptors have non-overlapping roles in responses to ethylene. Thus, differential control of receptor levels is likely to provide another mechanism to control specific responses to ethylene. This seems to be the case for the LeETR4 and LeETR6 receptors that have a particularly important role in controlling tomato fruit ripening and whose levels are controlled posttranscriptionally [90,91].

8. Conclusions and future directions A more refined model is emerging about the structure and function of the ethylene receptors and signal transduction pathway. This includes more information about the ethylene binding domain, physical interactions that occur between the receptors and down-stream components, and a more nuanced assessment of the unique roles that each isoform plays. However, even with these advances many fundamental questions remain regarding the ethylene receptors. For instance, while a great deal is now understood about the binding of ethylene to the receptors, the structure of the binding pocket and how the binding event is transduced through the receptor remains obscure. Additionally, there is a gap in our knowledge regarding the functional output of the receptors that modulates CTR1 activity. In this regard, one basic question that still needs to be answered is what effect does ethylene binding have on the receptor’s kinase activity? Also, while it is generally believed that the dimer represents the basic functional unit of the ethylene receptor, it is unclear whether or not higher order receptor clusters form to modulate receptor output. Despite these unanswered questions, it is clear that receptor levels affect ethylene-sensitivity. It is now apparent that plants have elaborate mechanisms at the level of the receptors to modulate various responses to ethylene since the receptor isoforms are not entirely redundant in function and there is differential, post-transcriptional regulation of the levels of particular receptor isoforms. This coupled with differences observed between species highlights the complexities and challenges of fully understanding ethylene signalling and receptor function. Acknowledgements The author apologizes to any researchers whose work was not included through oversight or space limitations. Thanks to Eric Schaller for helpful conversations and to Filippo de Franceschi and Matthew Christians for feedback on this manuscript. The author also thanks Sara Patterson for advice and support. References [1] A.K. Mattoo, J.C. Suttle, The Plant Hormone Ethylene, CRC Press, Inc., Boca Raton, 1991, pp. 1–337. [2] F. Abeles, P. Morgan, M.J. Saltveit, Ethylene in Plant Biology, Academic Press, San Diego, CA, 1992, pp. 1–414. [3] D. Neljubov, Uber die horizontale Nutation der Stengel von Pisum sativum und einiger Anderer, Pflanzen Beih. Bot. Zentralb. 10 (1901) 128–139. [4] E.C. Sisler, Measurement of ethylene binding in plant tissue, Plant Physiol. 64 (1979) 538–542. [5] E.C. Sisler, Partial purification of an ethylene-binding component from plant tissue, Plant Physiol. 66 (1980) 404–406. [6] D.E. Evans, T. Bengochea, A.J. Cairns, J.H. Dodds, M.A. Hall, Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L.: subcellular localization, Plant Cell Environ. 5 (1982) 101–107.

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