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Ethylene is a gaseous phytohormone and the first of this hormone class to be discovered. It is the simplest olefin gas and is biosynthesized by plants to regulate plant development, growth, and stress responses via a well-studied signaling pathway. One of the earliest reported responses to ethylene is the triple response. This response is common in eudicot seedlings grown in the dark and is characterized by reduced growth of the root and hypocotyl, an exaggerated apical hook, and a thickening of the hypocotyl. This proved a useful assay for genetic screens and enabled the identification of many components of the ethylene-signaling pathway. These components include a family of ethylene receptors in the membrane of the endoplasmic reticulum (ER); a protein kinase, called constitutive triple response 1 (CTR1); an ER-localized transmembrane protein of unknown biochemical activity, called ethylene-insensitive 2 (EIN2); and transcription factors such as EIN3, EIN3-like (EIL), and ethylene response factors (ERFs). These studies led to a linear model, according to which in the absence of ethylene, its cognate receptors signal to CTR1, which inhibits EIN2 and prevents downstream signaling. Ethylene acts as an inverse agonist by inhibiting its receptors, resulting in lower CTR1 activity, which releases EIN2 inhibition. EIN2 alters transcription and translation, leading to most ethylene responses. Although this canonical pathway is the predominant signaling cascade, alternative pathways also affect ethylene responses. This review summarizes our current understanding of ethylene signaling, including these alternative pathways, and discusses how ethylene signaling has been manipulated for agricultural and horticultural applications.
). It is biosynthesized by plants and is well-known to affect various developmental processes, such as seed germination, fruit ripening, senescence, and abscission, as well as responses to various stresses, such as flooding, high salt, and soil compaction (
). The ethylene signal transduction pathway has been extensively studied, in part because ethylene affects so many traits related to plant vigor and post-harvest physiology and storage.
Once biosynthesized, ethylene diffuses throughout the plant and binds to ethylene receptors to stimulate ethylene responses. It can also diffuse to surrounding plants and is the basis of the saying one bad apple spoils the bunch, where ethylene produced by an apple hastens the ripening of bananas. The ethylene-signaling pathway was predominantly delineated with research on Arabidopsis thaliana and is comprised of a combination of components that is not found in other pathways. This review will mainly focus on this research using Arabidopsis. However, it is worth pointing out that similar signaling pathways occur in diverse plants (
) so that information from Arabidopsis about ethylene signaling is usually applicable to other species.
Early molecular genetic studies uncovered several key components for ethylene signaling, including a family of receptors; the CTR1 protein kinase; EIN2, which is a transmembrane protein of unknown biochemical activity; and transcription factors, such as EIN3, EILs, and ERFs. This led to a linear, genetic model where, in the absence of ethylene, the receptors activate CTR1, which negatively regulates downstream signaling (Fig. 1). Ethylene functions as an inverse agonist by inhibiting the receptors, leading to release of inhibition by CTR1, resulting in ethylene responses (
). This genetic model provided a general framework that has been refined with further research, resulting in a more complete and detailed model for ethylene signaling, including surprising cases of cross-talk from the receptors to other signaling pathways, details for how a signal perceived at the ER membrane affects transcription in the nucleus, and multiple roles for EIN2. Details from this research have led to various ways to control ethylene signaling. Most of these controls are geared toward inhibiting ethylene responses to prevent post-harvest spoilage. However, there is also a need for stimulating ethylene responses, such as to cause premature germination of parasitic plants so that fields can be cleared of these problematic plants. These discoveries and applications will be summarized in this review.
Figure 1Simple genetic model of ethylene signaling. In black is shown a model for ethylene signaling based on molecular genetic experiments in Arabidopsis. These experiments showed that ethylene signaling involves ethylene receptors (ETR1, ERS1, ETR2, EIN4, and ERS2), the protein kinase CTR1, and EIN2 that signals to the transcription factors EIN3, EIL1, and EIL2. These, in turn, signal to other transcription factors, such as the ERFs, leading to ethylene responses. This has long been considered the canonical signaling pathway. In this model, CTR1 is a negative regulator of signaling. Ethylene functions as an inverse agonist, where it inhibits the receptors, which leads to lower activity of CTR1 releasing downstream components from inhibition by CTR1. More recent evidence has shown the existence of an alternative, “noncanonical” pathway (in gray), where ETR1 signals to histidine-containing AHPs and then to ARRs to modulate responses to ethylene.
Ethylene-signaling components and the canonical pathway
The first step in ethylene perception is the binding of ethylene to receptors. Ethylene receptors have homology to bacterial two-component receptors that signal via autophosphorylation on a histidine residue followed by phosphotransfer to an aspartate residue in the receiver domain of a response regulator protein (
). Ethylene receptors, as well as other two-component-like receptors, such as the phytochromes and cytokinin receptors, are believed to have been acquired by plants from the cyanobacterium that gave rise to chloroplasts (
Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus.
Proc. Natl. Acad. Sci. U.S.A.2002; 99 (12218172): 12246-12251
). It is thus interesting to note that ethylene binding has been observed in diverse cyanobacteria, and at least one cyanobacterium, Synechocystis, has a functional ethylene receptor that regulates cell surface properties to affect biofilm formation and phototaxis (
). Additionally, ethylene-binding affinities to some of these cyanobacteria and the heterologously expressed Synechocystis ethylene receptor are similar to what has been observed in plants (
), showing a conservation of this domain between these organisms. However, the organism where ethylene receptors first arose remains unknown. The observation that genes encoding for proteins with putative ethylene-binding domains are found in other phyla of bacteria (
By contrast, as will be discussed in more detail below, even though some of the plant ethylene receptor isoforms have retained histidine kinase activity, this activity is not crucial for ethylene perception. This is in contrast to the one cyanobacterial system so far characterized where phosphotransfer is central to the function of the receptor (
Novel photosensory two-component system (PixA-NixB-NixC) involved in the regulation of positive and negative phototaxis of cyanobacterium Synechocystis sp. PCC 6803.
). Additionally, some plant ethylene receptor isoforms have serine/threonine kinase activity, indicating that the outputs of these receptors in plants are now diverged from the ancestral proteins. Recent reviews present more information about ethylene receptors in nonplant species (
A study of the subcellular localisation of an ethylene binding site in developing cotyledons of Phaseolus vulgaris L. by high resolution autoradiography.
). In Arabidopsis, five isoforms have been identified and are referred to as ethylene response 1 (ETR1), ethylene response sensor 1 (ERS1), ETR2, ERS2, and EIN4 (
). There are also some mutations in these receptors that have no effect on ethylene binding but prevent signaling through the receptor, which also leads to ethylene insensitivity (
The different receptor isoforms in plants have similar domain architecture (Fig. 2) with three transmembrane α-helices at the N terminus, which comprises the ethylene-binding domain, followed by a GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) and kinase domain. Three of the five receptors also contain a receiver domain that is similar to what is found in bacterial two-component receptors (
). The subfamily 2 receptors contain additional amino acids at the N terminus that are unknown in function. The receptors can be further distinguished by their kinase activity. ETR1 has histidine kinase activity, whereas ETR2, ERS2, and EIN4 have serine/threonine kinase activity, and ERS1 has been documented to have both, depending on assay conditions, although it is believed to be a serine/threonine kinase in vivo (
Figure 2Diagram of domains of receptor isoforms. The receptors are dimers located in the ER membrane. Each dimer is stabilized by two disulfide bonds near the N terminus. All of the receptors contain transmembrane helices that comprise the ethylene-binding domain followed by a GAF and kinase domain. ETR1 is a histidine kinase, and the other four isoforms are serine/threonine kinases. Three of the five contain a receiver domain at the C terminus of the protein. The models for the receptors are based on published structural and computational studies on ETR1 (
), where each monomer coordinates a copper ion required for ethylene binding. In ETR1, the DHp domain of the kinase dimerizes, and a flexible region allows each kinase catalytic domain to associate with the DHp domain. It is unknown whether the kinase domains of the other isoforms also dimerize. The receiver domains are predicted to be orientated away from the central axis of the receptor dimer.
). In ETR1, it is thought that dimerization between monomers also occurs between the dimerization and histidine phosphotransfer (DHp) domains of each kinase domain (
). It is unclear whether dimerization between kinase domains of the other receptor isoforms occurs. It has also been suggested that heterodimers are possible (
Analysis of ethylene signal-transduction kinetics associated with seedling-growth response and chitinase induction in wild-type and mutant Arabidopsis.
). One difference between heterologously expressed receptors and those in planta is that ethylene dissociates from the former with a single, slow rate having a half-time of release of ∼10–12 h (
). In planta, there is an initial, rapid release of ethylene in the first 30 min after ethylene removal, followed by slow release with similar kinetics to the heterologously expressed receptors. Because ethylene can enhance the proteolysis of ethylene receptors (
). This has led to a model of the ETR1 dimer where the DHp domain of the histidine kinase domain dimerizes with the DHp of the other monomer (Fig. 2). In this model, the catalytic domain associates with the DHp domain. The catalytic and receiver domains are modeled to extend outward from the DHp pair. The orientation of the receiver domain in relationship to the remainder of the protein is predicted to be different from prokaryotic histidine kinases, suggesting that this domain may be diverged in function from prokaryotes (
). Additionally, structural studies show that the γ-loop of ETR1, which is part of the catalytic region of receiver domains, is in a different orientation from characterized prokaryote receiver domains (
) suggests that ethylene binds in the middle of helices 1 and 2 and the signal is transduced via helix 3. The mechanistic details of this transduction through the receptor are unknown.
A key issue in ethylene signaling has been to determine how proteins bind ethylene with high affinity, and mutational studies have identified amino acids in helices 1 and 2 that are important for ethylene binding (
). Cys-65 in helix 2 is required for coordination of copper because the etr1-1 mutant receptor with a C65Y mutation is unable to bind copper or ethylene (
). Mutants such as this render the plant ethylene-insensitive. Additionally, several studies have determined that the ER membrane–localized copper transporter, responsive to antagonist 1 (RAN1), physically interacts with at least some of the receptors and is needed for delivery of copper and proper biogenesis of the ethylene receptors (
). Recent experimental evidence, however, indicates that there are two copper ions per receptor dimer that are modeled to be coordinated by amino acids in helices 1 and 2 of each monomer (
The biochemical output of the receptors has yet to be determined. The GAF, kinase, and receiver domains are the likely output domains, but the specifics of how ethylene signal is transduced are unknown. This is complicated by research showing that even though the receptors have overlapping roles for many traits, for specific traits or under specific conditions, individual receptor isoforms have a role, whereas others do not (
). In some cases, individual isoforms display opposite roles from other isoforms. For instance, ETR1 is necessary and sufficient for ethylene-stimulated nutational bending of hypocotyls in dark-grown Arabidopsis seedlings, whereas the other four receptor isoforms inhibit this response (
). Also, loss of ETR1, and to a lesser extent EIN4, results in plants that are less sensitive to the plant hormone abscisic acid (ABA) during seed germination, whereas loss of ETR2 causes plants to be more sensitive to ABA (
). There is recent evidence that ETR1 and ETR2 are signaling independently of CTR1 to cause the changes in ABA responsiveness, but the exact pathway has yet to be determined (
). These observations indicate that there are likely to be differences in the biochemical output between receptor isoforms. Although some of these differences may arise from different kinase specificities (
), this does not easily explain all of these differences.
Ethylene receptors are homologous to bacterial two-component receptors. The simplest bacterial two-component system signals by histidine autophosphorylation followed by relay of the phosphoryl to a conserved aspartate on a receiver domain of a response regulator protein, although more complex variations of this exist (
). Similarly, receptor serine/threonine kinase activity does not appear to be required for ethylene responses but may have a modulatory role in ethylene receptor signal transduction and responses (
Complexes of receptor dimers have been proposed to explain the large range of ethylene concentrations that plants respond to and to explain how one mutant receptor might affect other, nonmutant receptors (
). Receptor dimer clusters are proposed as a way for signal amplification to occur, much like how bacterial chemoreceptors function. In chemoreceptors, ligand binding to one receptor dimer can affect the signaling state of neighboring, unbound receptor dimers to increase signal output (
). Specific proteins have been identified as interacting partners with all or a subset of the ethylene receptors. This includes interactions with RAN1 that may be important for correct delivery of copper to the receptors (
). Other interacting partners are less characterized. Reversion to ethylene sensitivity 1 (RTE1) interacts with ETR1 and tetratricopeptide repeat protein 1 (TRP1) with ERS1 to modulate signaling (
SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development.
). EIN2 is required for ethylene signaling and is part of the NRAMP (natural resistance-associated microphage protein) family of metal transporters; it is comprised of a large, N-terminal portion containing multiple transmembrane domains in the ER membrane and a cytosolic C-terminal portion (
). In the case of ETR1, the kinase domain of the receptor is required for interactions with both CTR1 and EIN2, although ETR1 histidine kinase activity is only important for modulating interactions with EIN2 (
). These physical interactions appear to be important because mutations in CTR1 that abolish receptor-CTR1 interactions result in a nonfunctional CTR1 (
), which may result in EIN2 ubiquitination via an Skp1 Cullen F-box (SCF) E3 ubiquitin ligase complex containing the EIN2-targeting protein 1 (ETP1) and ETP2 F-box proteins and subsequent proteolysis by the 26S proteasome (
). A downstream consequence of this is that the EIN3, EIL1, and EIL2 transcription factors are targeted for ubiquitination by an SCF E3 complex that contains the EBF1 and EBF2 F-box proteins (
Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis.
). The breakdown of these transcription factors prevents ethylene responses. Thus, in the absence of ethylene, signal transduction in the pathway is blocked because EIN2 levels are low.
Figure 3Model for ethylene signaling. RAN1 is a copper transporter that delivers copper to the lumen of the ER, where it is required for the biogenesis of the receptors and is used as a cofactor by the receptors to bind ethylene. A, in the absence of ethylene, the receptors signal to CTR1, which phosphorylates EIN2. This results in the ubiquitination of EIN2 by an SCF E3 containing the ETP1/2 F-box proteins, leading to EIN2 degradation by the proteasome. Because EIN2 levels are low, an SCF-E3 containing the EBF1/2 F-box proteins ubiquitinates EIN3 and EIL1, leading to their degradation by the proteasome and preventing them from affecting transcription in the nucleus. B, in the presence ethylene, the receptors bind ethylene via a copper cofactor. The binding of ethylene is modeled to cause a conformational change that either reduces CTR1 kinase activity or, as shown, results in CTR1 being sequestered by the receptors so that CTR1 can no longer phosphorylate EIN2. The reduction in EIN2 phosphorylation results in less EIN2 ubiquitination and an increase in EIN2 levels. An unknown protease cleaves EIN2, releasing the C-terminal end (EIN2-C) from the N-terminal end (EIN2-N). One fate of EIN2-C is to bind the RNAs for EBF1 and EBF2 and become sequestered in processing bodies (P-bodies). The reduction of EBF1/2 results in less ubiquitination of EIN3 and EIL1, causing higher EIN3/EIL1 levels. The other fate of EIN2-C is to translocate to the nucleus, where it increases the transcriptional activity of EIN3/EIL1 via ENAP1. This leads to numerous transcriptional changes. In parallel with this pathway, phosphoryl transfer from a conserved histidine in the ETR1 DHp domain to an aspartate in the receiver domain occurs. This is followed by phosphoryl transfer from this residue to AHPs and finally ARRs resulting in transcriptional changes.
In the presence of ethylene, the receptors are inhibited, leading to less phosphorylation of EIN2 by CTR1. Genetic data predict that the binding of ethylene to the receptors should reduce the catalytic activity of CTR1. However, this has not yet been directly tested. Ethylene enhances the interaction between ETR1 and both CTR1 and EIN2 (
). Thus, an alternative explanation for reduced EIN2 phosphorylation by CTR1 is that the binding of ethylene to the receptors results in conformational changes in the receptors that reduces the physical interaction between CTR1 and EIN2, leading to less EIN2 phosphorylation. It is thought that when EIN2 phosphorylation is reduced, there is less EIN2 ubiquitination, resulting in an increase in EIN2 levels and subsequent cleavage of EIN2 by an unknown protease to release the C-terminal portion of EIN2 (EIN2-C) from the membrane-bound N-terminal (EIN2-N) portion (
The role of EIN2-N is unknown, but it has diverged from other NRAMP proteins, because no metal transport activity has been detected in heterologously expressed EIN2 and it cannot rescue yeast deficient in metal uptake (
). However, there are hints that EIN2-N has a role in ethylene signaling. In rice, mao huzi 3 (mhz3) mutants are ethylene-insensitive, and the MHZ3 protein physically interacts with OsEIN2-N and regulates OsEIN2 abundance; similar genes have been identified in Arabidopsis that affect ethylene signaling (
). These data indicate the need to further study EIN2-N to delineate the mechanism by which it affects ethylene signaling.
By contrast, EIN2-C has two known roles. One is to bind the mRNAs that encode for EBF1 and EBF2, whereupon this protein/RNA complex associates with processing bodies (
). This results in the degradation of these mRNAs by exoribonuclease 4 (XRN4, also known as EIN5), which is a 5′ → 3′ exoribonuclease known to affect ethylene signaling (
). A consequence of the degradation of EBF1 and EBF2 mRNA is that degradation of EIN3 and EIL1 and probably EIL2 is reduced, leading to more ethylene signaling (
Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis.
). EIN2-C also contains a nuclear localization sequence (NLS). EIN2-C diffuses into the nucleus, where it associates with EIN2 nuclear associated protein 1 (ENAP1), which is required for the ability of EIN2-C to regulate EIN3-dependent transcription (
). Thus EIN2-C provides both transcriptional and translational control to regulate EIN3 and the related EIL1 transcription factor to cause most ethylene responses. This is supported by a recent study where ethylene-stimulated changes in the metabolome did not always correlate with changes in the transcriptome (
). Thus, there are more functions for EIN2 that have yet to be discovered.
The increase in EIN3, EIL1, and EIL2 activity caused by EIN2-C leads to changes in the transcription of other ethylene response genes, including other transcription factors, such as the ERFs (
). Recent studies have identified histone modifications as having a role in this transcriptional control. Mutational experiments revealed that several histone acetyltransferases and histone deacetylases affect ethylene signaling (
). Even though more details about transcriptional regulation are being discovered, it is also clear from a recent metabolome study that changes in metabolism occur in response to ethylene that are not predicted by changes in the transcriptome (
). This indicates that there is additional regulation for responses to this hormone.
In summary, the model for the canonical ethylene-signaling pathway has developed from a simple genetic model to a more complex model with many more biochemical details. However, there are still gaps in our understanding of this signal transduction pathway.
Noncanonical signaling
The model discussed above is largely linear, and it summarizes the main pathway by which ethylene affects plants. Nonetheless, it is clear from diverse studies that the ethylene-signaling pathway involves feed-forward and feedback regulation leading to sensitization and adaptation (
Maize and Arabidopsis ARGOS proteins interact with ethylene receptor signaling complex, supporting a regulatory role for ARGOS in ethylene signal transduction.
Dominant gain-of-function mutations in transmembrane domain III of ERS1 and ETR1 suggest a novel role for this domain in regulating the magnitude of ethylene response in Arabidopsis.
Mutational loss of the prohibitin AtPHB3 results in an extreme constitutive ethylene response phenotype coupled with partial loss of ethylene-inducible gene expression in Arabidopsis seedlings.
The eer5 mutation, which affects a novel proteasome-related subunit, indicates a prominent role for the COP9 signalosome in resetting the ethylene-signaling pathway in Arabidopsis.
). Most of this research has identified adaptation mechanisms at the level of the receptors. For instance, the levels of the receptors themselves can regulate sensitivity, where higher levels lead to less sensitivity and lower levels to more sensitivity (
The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family.
Proc. Natl. Acad. Sci. U.S.A.2000; 97 (10792050): 5663-5668
Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent.
). However, it is also now clear that other proteins affect sensitivity at the levels of the receptors. This includes negative regulation by RTE1 and the family of proteins called auxin-regulated gene involved in organ size (ARGOS) (
The existence of nonlinear components to what has been considered the canonical pathway raises the possibility that other ethylene-signaling pathways exist outside of or as branch points from this core pathway. This is an area of active research, and in the cases discussed below, evidence is provided showing that signaling occurs, at least in part, via components not contained in the canonical pathway presented above. These alternative (noncanonical) pathways are not necessary for ethylene responses but appear to have roles in modulating responses to ethylene or in altering responses to other hormones.
Results from several studies have led to the suggestion that the ethylene receptors signal independently of CTR1 or EIN2 (
). For instance, epistasis analysis has shown that the role of ETR1 and ETR2 in the control of seed germination by ABA is, at least in part, independent of CTR1 (
). It is possible that such alternative signaling occurs via CTR1 homologues, but so far no CTR1 homologue has been identified as being involved in this. Even though ETR1 histidine kinase activity is not required for ethylene signaling, this activity does modulate sensitivity to ethylene, growth recovery kinetics when ethylene is removed, growth of root apical meristem, seed germination under stress conditions or in response to ABA, and interactions with EIN2 (
). Likely targets for phosphorelay from ETR1 are components of the cytokinin signaling pathway (Fig. 1). The cytokinin receptors are two-component receptors in plants that, unlike the ethylene receptors, use phosphorelay as the primary route for signaling (
). In this pathway, the phosphoryl is transferred from the cytokinin receptors to histidine-containing phosphotransfer proteins (AHP family in Arabidopsis) and finally to response regulator proteins (ARR family in Arabidopsis) that function as transcription factors. Various studies have demonstrated that ETR1 physically interacts with ARR and AHP proteins (
). The affinity between ETR1 and AHP1 is altered by their phosphorylation state, where it is highest if one protein is phosphorylated and the other is not (
In support of interactions between ETR1 and the cytokinin pathway having functional consequences, mutational analyses revealed that the ARRs are involved in ethylene responses such as sensitivity to ethylene, recovery kinetics after ethylene is removed, stomatal aperture control, and the regulation of root apical meristem (
). Similarly, null mutants in several AHPs and ARRs prolong growth recovery when ethylene is removed, similar to what is observed in plants deficient in ETR1 histidine kinase activity (
). Together, these results are consistent with a model where ETR1 histidine kinase activity is directly involved in affecting components of the cytokinin pathway, resulting in changes in transcription that modulate ethylene responses (Fig. 3). There is some overlap between transcriptional changes caused by ethylene and cytokinin (
), raising the possibility that there are both overlapping and nonoverlapping targets of transcriptional control from this signaling pathway involving ETR1 histidine kinase and the well-known pathway involving EIN3 and EILs. It is interesting to note that in rice, a histidine kinase (MHZ1/OsHK1) that may have a role in cytokinin signaling functions downstream of the OsERS2 ethylene receptor and signals independently of OsEIN2 (
). Thus, our model for canonical ethylene signaling probably needs to be expanded to include secondary pathways such as phosphorelay from some of the ethylene receptors to the AHPs and ARRs.
It should be noted that biochemical experiments show that ETR1 histidine autophosphorylation decreases upon binding of ethylene or ethylene receptor agonists (
). Histidine kinases can carry out multiple enzymatic reactions, including kinase, phosphatase, and phosphotransfer reactions, and receiver domains can catalyze both phosphotransfer and autodephosphorylation reactions (
). Given this complexity, one possible resolution to this discrepancy between biochemical and genetic data is that histidine autophosphorylation occurs in the absence of ethylene, but phosphotransfer to the receiver domain does not
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