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Ethylene signaling in plants

Open AccessPublished:April 24, 2020DOI:https://doi.org/10.1074/jbc.REV120.010854
      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.

      Introduction

      Ethylene (IUPAC name ethene) is the simplest olefin gas and was the first gaseous molecule shown to function as a hormone (
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      History of research on the plant hormone ethylene.
      ). 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 (
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      ). 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 (
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      ) 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 (
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      ). 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 thumbnail gr1
      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 (
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      ). 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 (
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      Evidence for a plastid origin of plant ethylene receptor genes.
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      ). Data from a recent phylogenetic analysis suggest a common origin for the ethylene-binding domain in cyanobacteria and plants (
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      ). 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 (
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      Ethylene regulates the physiology of the cyanobacterium Synechocystis sp. PCC 6803 via an ethylene receptor.
      ). 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 (
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      ), 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 (
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      Ethylene regulates the physiology of the cyanobacterium Synechocystis sp. PCC 6803 via an ethylene receptor.
      ) will make answering this question difficult.
      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 (
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      ). 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 (
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      Plants contain multiple ethylene receptor isoforms. Early studies identified ethylene-binding sites in the ER membranes of plants (
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      ), and subsequent research on specific receptor isoforms from various plants confirmed that ethylene receptors are localized to the ER (
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      ). 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 (
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      ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis.
      ). Mutations in any one of these receptors that prevent ethylene binding lead to an ethylene-insensitive plant (
      • Hall A.E.
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      ,
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      • Somerville C.
      • Kende H.
      Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana.
      ,
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      Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators.
      ,
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      ). 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 (
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      Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis.
      ).
      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 (
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      ,
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      Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).
      ). The receptors fall into two subfamilies with ETR1 and ERS1 in subfamily 1 and the other three isoforms in subfamily 2 (
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      ). 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 (
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      ).
      Figure thumbnail gr2
      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 (
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      Structural model of the ETR1 ethylene receptor transmembrane sensor domain.
      ), 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.
      The receptors form homodimers that are stabilized at their N termini by two disulfide bonds (
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      ). Nevertheless, these disulfide bonds are necessary neither for binding of ethylene to ETR1 (
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      Ethylene receptors function as components of high-molecular-mass protein complexes in Arabidopsis.
      ) nor for a functional ETR1 receptor in planta (
      • Xie F.
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      Receptor signal output mediated by the ETR1 N-terminus is primarily subfamily I receptor dependent.
      ). In ETR1, it is thought that dimerization between monomers also occurs between the dimerization and histidine phosphotransfer (DHp) domains of each kinase domain (
      • Mayerhofer H.
      • Panneerselvam S.
      • Kaljunen H.
      • Tuukkanen A.
      • Mertens H.D.T.
      • Mueller-Dieckmann J.
      Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).
      ). It is unclear whether dimerization between kinase domains of the other receptor isoforms occurs. It has also been suggested that heterodimers are possible (
      • Grefen C.
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      • Obrdlik P.
      • Harter K.
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      Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members.
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      Molecular analysis of protein-protein interactions in the ethylene pathway in the different ethylene receptor subfamilies.
      ). Evidence that these are receptors is that all of these proteins bind ethylene with high affinity (
      • Schaller G.E.
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      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ,
      • Hall A.E.
      • Findell J.L.
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      • Sisler E.C.
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      Ethylene perception by the ERS1 protein in Arabidopsis.
      ,
      • O'Malley R.C.
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      Ethylene-binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato.
      ,
      • McDaniel B.K.
      • Binder B.M.
      Ethylene receptor 1 (ETR1) is sufficient and has the predominant role in mediating inhibition of ethylene responses by silver in Arabidopsis thaliana.
      ), and specific mutations in any one of these proteins lead to ethylene insensitivity (
      • Bleecker A.B.
      • Estelle M.A.
      • Somerville C.
      • Kende H.
      Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana.
      ,
      • Hua J.
      • Chang C.
      • Sun Q.
      • Meyerowitz E.M.
      Ethylene insensitivity conferred by Arabidopsis ERS gene.
      ,
      • Hua J.
      • Sakai H.
      • Nourizadeh S.
      • Chen Q.G.
      • Bleecker A.B.
      • Ecker J.R.
      • Meyerowitz E.M.
      EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.
      ,
      • Sakai H.
      • Hua J.
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      • Chang C.
      • Medrano L.J.
      • Bleecker A.B.
      • Meyerowitz E.M.
      ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis.
      ,
      • Chen Q.G.
      • Bleecker A.B.
      Analysis of ethylene signal-transduction kinetics associated with seedling-growth response and chitinase induction in wild-type and mutant Arabidopsis.
      ). Similar proteins from tomato also bind ethylene with high affinity and when mutated lead to ethylene insensitivity (
      • O'Malley R.C.
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      • O'Donnell P.
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      Ethylene-binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato.
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      An ethylene-inducible component of signal transduction encoded by Never-ripe.
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      Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life.
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      Role of SlETR7, a newly discovered ethylene receptor, in tomato plant and fruit development.
      ).
      Ethylene binds to the N-terminal, transmembrane portion of heterologously expressed receptors with Kd values reported in the nanomolar range (
      • Rodríguez F.I.
      • Esch J.J.
      • Hall A.E.
      • Binder B.M.
      • Schaller G.E.
      • Bleecker A.B.
      A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
      ,
      • Schaller G.E.
      • Bleecker A.B.
      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ,
      • McDaniel B.K.
      • Binder B.M.
      Ethylene receptor 1 (ETR1) is sufficient and has the predominant role in mediating inhibition of ethylene responses by silver in Arabidopsis thaliana.
      ), which corresponds to ethylene-binding affinities reported in plants (
      • Sisler E.C.
      Measurement of ethylene binding in plant tissue.
      ,
      • Blankenship S.M.
      • Sisler E.C.
      Ethylene binding changes in apple and morning glory during ripening and senescence.
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      Ethylene binding site affinity in ripening apples.
      ,
      • Sanders I.O.
      • Ishizawa K.
      • Smith A.R.
      • Hall M.A.
      Ethylene binding and action in rice seedlings.
      ,
      • Smith A.R.
      • Robertson D.
      • Sanders I.O.
      • Williams R.A.N.
      • Hall M.A.
      Ethylene binding sites.
      ,
      • Sisler E.C.
      • Reid M.S.
      • Yang S.F.
      Effect of antagonists of ethylene action on binding of ethylene in cut carnations.
      ,
      • Goren R.
      • Sisler E.C.
      Ethylene-binding characteristics in phaseolus, citrus, and ligustrum plants.
      ,
      • Sanders I.O.
      • Harpham N.V.J.
      • Raskin I.
      • Smith A.R.
      • Hall M.A.
      Ethylene binding in wild type and mutant Arabidopsis thaliana (L.) Heynh.
      ). 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 (
      • Schaller G.E.
      • Bleecker A.B.
      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ,
      • O'Malley R.C.
      • Rodriguez F.I.
      • Esch J.J.
      • Binder B.M.
      • O'Donnell P.
      • Klee H.J.
      • Bleecker A.B.
      Ethylene-binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato.
      ,
      • McDaniel B.K.
      • Binder B.M.
      Ethylene receptor 1 (ETR1) is sufficient and has the predominant role in mediating inhibition of ethylene responses by silver in Arabidopsis thaliana.
      ), whereas there are two rate constants of release in planta (
      • Sanders I.O.
      • Harpham N.V.J.
      • Raskin I.
      • Smith A.R.
      • Hall M.A.
      Ethylene binding in wild type and mutant Arabidopsis thaliana (L.) Heynh.
      ,
      • Sisler E.C.
      Ethylene-binding components in plants.
      ). 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 (
      • Chen Y.-F.
      • Shakeel S.N.
      • Bowers J.
      • Zhao X.-C.
      • Etheridge N.
      • Schaller G.E.
      Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis.
      ,
      • Shakeel S.N.
      • Gao Z.
      • Amir M.
      • Chen Y.-F.
      • Rai M.I.
      • Haq N.U.
      • Schaller G.E.
      Ethylene regulates levels of ethylene receptor/CTR1 signaling complexes in Arabidopsis thaliana.
      ,
      • Kevany B.M.
      • Tieman D.M.
      • Taylor M.G.
      • Cin V.D.
      • Klee H.J.
      Ethylene receptor degradation controls the timing of ripening in tomato fruit.
      ), this rapid release of ethylene from receptors in plants is likely due to proteolysis of the ethylene-bound receptors.
      The cytosolic domains of ETR1 have been structurally characterized (
      • Müller-Dieckmann H.-J.
      • Grantz A.A.
      • Kim S.-H.
      The structure of the signal receiver domain of the Arabidopsis thaliana ethylene receptor ETR1.
      ,
      • Mayerhofer H.
      • Panneerselvam S.
      • Kaljunen H.
      • Tuukkanen A.
      • Mertens H.D.T.
      • Mueller-Dieckmann J.
      Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).
      ,
      • Hung Y.-L.
      • Jiang I.
      • Lee Y.-Z.
      • Wen C.-K.
      • Sue S.-C.
      NMR study reveals the receiver domain of Arabidopsis ETHYLENE RESPONSE1 ethylene receptor as an atypical type response regulator.
      ). 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 (
      • Hung Y.-L.
      • Jiang I.
      • Lee Y.-Z.
      • Wen C.-K.
      • Sue S.-C.
      NMR study reveals the receiver domain of Arabidopsis ETHYLENE RESPONSE1 ethylene receptor as an atypical type response regulator.
      ). 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 (
      • Müller-Dieckmann H.-J.
      • Grantz A.A.
      • Kim S.-H.
      The structure of the signal receiver domain of the Arabidopsis thaliana ethylene receptor ETR1.
      ,
      • Hung Y.-L.
      • Jiang I.
      • Lee Y.-Z.
      • Wen C.-K.
      • Sue S.-C.
      NMR study reveals the receiver domain of Arabidopsis ETHYLENE RESPONSE1 ethylene receptor as an atypical type response regulator.
      ). No structural information is published characterizing the ethylene-binding domain, but a computational model is available (
      • Schott-Verdugo S.
      • Müller L.
      • Classen E.
      • Gohlke H.
      • Groth G.
      Structural model of the ETR1 ethylene receptor transmembrane sensor domain.
      ). This study coupled with prior research (
      • Wang W.
      • Esch J.J.
      • Shiu S.H.
      • Agula H.
      • Binder B.M.
      • Chang C.
      • Patterson S.E.
      • Bleecker A.B.
      Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis.
      ) 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 (
      • Wang W.
      • Esch J.J.
      • Shiu S.H.
      • Agula H.
      • Binder B.M.
      • Chang C.
      • Patterson S.E.
      • Bleecker A.B.
      Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis.
      ,
      • Rodríguez F.I.
      • Esch J.J.
      • Hall A.E.
      • Binder B.M.
      • Schaller G.E.
      • Bleecker A.B.
      A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
      ,
      • Schaller G.E.
      • Bleecker A.B.
      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ). Based on olefin chemistry, several transition metals were initially suggested as cofactors for binding activity (
      • Thompson J.
      • Harlow R.
      • Whitney J.
      Copper(I)-olefin complexes: support for the proposed role of copper in the ethylene effect in plants.
      ,
      • Burg S.P.
      • Burg E.A.
      Molecular requirements for the biological activity of ethylene.
      ,
      • Sisler E.C.
      Ethylene activity of some π-acceptor compounds.
      ,
      • Beyer E.M.
      A potent inhibitor of ethylene action in plants.
      ). It was later determined that ETR1 coordinates copper ions, which act as the cofactor for ethylene binding (
      • Rodríguez F.I.
      • Esch J.J.
      • Hall A.E.
      • Binder B.M.
      • Schaller G.E.
      • Bleecker A.B.
      A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
      ). 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 (
      • Rodríguez F.I.
      • Esch J.J.
      • Hall A.E.
      • Binder B.M.
      • Schaller G.E.
      • Bleecker A.B.
      A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
      ,
      • Bleecker A.B.
      • Estelle M.A.
      • Somerville C.
      • Kende H.
      Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana.
      ,
      • Chang C.
      • Kwok S.F.
      • Bleecker A.B.
      • Meyerowitz E.M.
      Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators.
      ,
      • Schaller G.E.
      • Bleecker A.B.
      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ). 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 (
      • Hirayama T.
      • Kieber J.J.
      • Hirayama N.
      • Kogan M.
      • Guzman P.
      • Nourizadeh S.
      • Alonso J.M.
      • Dailey W.P.
      • Dancis A.
      • Ecker J.R.
      RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.
      ,
      • Woeste K.E.
      • Kieber J.J.
      A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a Rosette-lethal phenotype.
      ,
      • Himelblau E.
      • Amasino R.M.
      Nutrients mobilized from leaves during leaf senescence.
      ,
      • Binder B.M.
      • Rodríguez F.I.
      • Bleecker A.B.
      The copper transporter RAN1 is essential for biogenesis of ethylene receptors in Arabidopsis.
      ,
      • Hoppen C.
      • Müller L.
      • Hänsch S.
      • Uzun B.
      • Milić D.
      • Meyer A.J.
      • Weidtkamp-Peters S.
      • Groth G.
      Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.
      ). Because copper co-purifies with the ETR1 dimer with a 1:1 stoichiometry, it was long thought that each receptor dimer contains one copper ion (
      • Rodríguez F.I.
      • Esch J.J.
      • Hall A.E.
      • Binder B.M.
      • Schaller G.E.
      • Bleecker A.B.
      A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
      ). 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 (
      • Schott-Verdugo S.
      • Müller L.
      • Classen E.
      • Gohlke H.
      • Groth G.
      Structural model of the ETR1 ethylene receptor transmembrane sensor domain.
      ).
      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 (
      • McDaniel B.K.
      • Binder B.M.
      Ethylene receptor 1 (ETR1) is sufficient and has the predominant role in mediating inhibition of ethylene responses by silver in Arabidopsis thaliana.
      ,
      • Liu Q.
      • Xu C.
      • Wen C.-K.
      Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis.
      ,
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Zutz T.C.
      • Bleecker A.B.
      Ethylene stimulates nutations that are dependent on the ETR1 receptor.
      ,
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Moore J.M.
      • Parks B.M.
      • Spalding E.P.
      • Bleecker A.B.
      Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis.
      ,
      • Wilson R.L.
      • Bakshi A.
      • Binder B.M.
      Loss of the ETR1 ethylene receptor reduces the inhibitory effect of far-red light and darkness on seed germination of Arabidopsis thaliana.
      ,
      • Wilson R.L.
      • Kim H.
      • Bakshi A.
      • Binder B.M.
      The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress.
      ,
      • Bakshi A.
      • Piya S.
      • Fernandez J.C.
      • Chervin C.
      • Hewezi T.
      • Binder B.M.
      Ethylene receptors signal via a non-canonical pathway to regulate abscisic acid responses.
      ,
      • Bakshi A.
      • Wilson R.L.
      • Lacey R.F.
      • Kim H.
      • Wuppalapati S.K.
      • Binder B.M.
      Identification of regions in the receiver domain of the ETHYLENE RESPONSE1 ethylene receptor of Arabidopsis important for functional divergence.
      ,
      • Kim H.
      • Helmbrecht E.E.
      • Stalans M.B.
      • Schmitt C.
      • Patel N.
      • Wen C.-K.
      • Wang W.
      • Binder B.M.
      Ethylene receptor ETR1 domain requirements for ethylene responses in Arabidopsis seedlings.
      ,
      • Harkey A.F.
      • Watkins J.M.
      • Olex A.L.
      • DiNapoli K.T.
      • Lewis D.R.
      • Fetrow J.S.
      • Binder B.M.
      • Muday G.K.
      Identification of transcriptional and receptor networks that control root responses to ethylene.
      ). 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 (
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Zutz T.C.
      • Bleecker A.B.
      Ethylene stimulates nutations that are dependent on the ETR1 receptor.
      ,
      • Kim H.
      • Helmbrecht E.E.
      • Stalans M.B.
      • Schmitt C.
      • Patel N.
      • Wen C.-K.
      • Wang W.
      • Binder B.M.
      Ethylene receptor ETR1 domain requirements for ethylene responses in Arabidopsis seedlings.
      ). 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 (
      • Wilson R.L.
      • Kim H.
      • Bakshi A.
      • Binder B.M.
      The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress.
      ,
      • Bakshi A.
      • Wilson R.L.
      • Lacey R.F.
      • Kim H.
      • Wuppalapati S.K.
      • Binder B.M.
      Identification of regions in the receiver domain of the ETHYLENE RESPONSE1 ethylene receptor of Arabidopsis important for functional divergence.
      ). 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 (
      • Bakshi A.
      • Piya S.
      • Fernandez J.C.
      • Chervin C.
      • Hewezi T.
      • Binder B.M.
      Ethylene receptors signal via a non-canonical pathway to regulate abscisic acid responses.
      ). 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 (
      • Gamble R.L.
      • Coonfield M.L.
      • Schaller G.E.
      Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis.
      ,
      • Moussatche P.
      • Klee H.J.
      Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family.
      ), 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 (
      • Gao R.
      • Stock A.M.
      Biological insights from structures of two-component proteins.
      ). Despite the fact that ETR1 possesses histidine kinase activity that is modulated by ethylene (
      • Gamble R.L.
      • Coonfield M.L.
      • Schaller G.E.
      Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis.
      ,
      • Moussatche P.
      • Klee H.J.
      Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family.
      ,
      • Voet-van-Vormizeele J.
      • Groth G.
      Ethylene controls autophosphorylation of the histidine kinase domain in ethylene receptor ETR1.
      ), this activity is not required for responses to ethylene (
      • Qu X.
      • Schaller G.E.
      Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.
      ,
      • Wang W.
      • Hall A.E.
      • O'Malley R.
      • Bleecker A.B.
      Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission.
      ). Rather, it may subtly modulate receptor signaling to downstream components (
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Moore J.M.
      • Parks B.M.
      • Spalding E.P.
      • Bleecker A.B.
      Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis.
      ,
      • Qu X.
      • Schaller G.E.
      Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.
      ,
      • Hall B.P.
      • Shakeel S.N.
      • Amir M.
      • Ul Haq N.
      • Qu X.
      • Schaller G.E.
      Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis.
      ,
      • Binder B.M.
      • Kim H.J.
      • Mathews D.E.
      • Hutchison C.E.
      • Kieber J.J.
      • Schaller G.E.
      A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene.
      ,
      • Street I.H.
      • Aman S.
      • Zubo Y.
      • Ramzan A.
      • Wang X.
      • Shakeel S.N.
      • Kieber J.J.
      • Schaller G.E.
      Ethylene inhibits cell proliferation of the Arabidopsis root meristem.
      ), including interactions with EIN2 (
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ). 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 (
      • Chen T.
      • Liu J.
      • Lei G.
      • Liu Y.-F.
      • Li Z.-G.
      • Tao J.-J.
      • Hao Y.-J.
      • Cao Y.-R.
      • Lin Q.
      • Zhang W.-K.
      • Ma B.
      • Chen S.-Y.
      • Zhang J.-S.
      Effects of tobacco ethylene receptor mutations on receptor kinase activity, plant growth and stress 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 (
      • Chen Y.-F.
      • Gao Z.
      • Kerris 3rd, R.J.
      • Wang W.
      • Binder B.M.
      • Schaller G.E.
      Ethylene receptors function as components of high-molecular-mass protein complexes in Arabidopsis.
      ,
      • Xie F.
      • Liu Q.
      • Wen C.-K.
      Receptor signal output mediated by the ETR1 N-terminus is primarily subfamily I receptor dependent.
      ,
      • Gao Z.
      • Wen C.-K.
      • Binder B.M.
      • Chen Y.-F.
      • Chang J.
      • Chiang Y.-H.
      • Kerris 3rd, R.J.
      • Chang C.
      • Schaller G.E.
      Heteromeric interactions among ethylene receptors mediate signaling in Arabidopsis.
      ,
      • Gamble R.L.
      • Qu X.
      • Schaller G.E.
      Mutational analysis of the ethylene receptor ETR1: role of the histidine kinase domain in dominant ethylene insensitivity.
      ,
      • Gao Z.
      • Schaller G.E.
      The role of receptor interactions in regulating ethylene signal transduction.
      ,
      • Binder B.M.
      • Bleecker A.B.
      A model for ethylene receptor function and 1-methylcyclopropene action.
      ,
      • Liu Q.
      • Wen C.-K.
      Arabidopsis ETR1ERS1 differentially repress the ethylene response in combination with other ethylene receptor genes.
      ). As an example, plants can respond to ethylene at levels down to 0.2 nl/liter (
      • Binder B.M.
      • Mortimore L.A.
      • Stepanova A.N.
      • Ecker J.R.
      • Bleecker A.B.
      Short-term growth responses to ethylene in Arabidopsis seedlings are EIN3/EIL1 independent.
      ), which is at least 300-fold below the Kd of binding to the receptors (
      • Schaller G.E.
      • Bleecker A.B.
      Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
      ). 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 (
      • Bray D.
      • Levin M.D.
      • Morton-Firth C.J.
      Receptor clustering as a cellular mechanism to control sensitivity.
      ,
      • Parkinson J.S.
      • Hazelbauer G.L.
      • Falke J.J.
      Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update.
      ). Structural studies suggest that CTR1 or the receptor receiver domains, or both, may be involved in the formation of ethylene receptor clusters (
      • Mayerhofer H.
      • Panneerselvam S.
      • Kaljunen H.
      • Tuukkanen A.
      • Mertens H.D.T.
      • Mueller-Dieckmann J.
      Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).
      ,
      • Mayerhofer H.
      • Panneerselvam S.
      • Mueller-Dieckmann J.
      Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk.
      ). It remains to be determined whether this is important in ethylene signaling.
      The receptors also form higher-order complexes with other proteins (
      • Chen Y.-F.
      • Gao Z.
      • Kerris 3rd, R.J.
      • Wang W.
      • Binder B.M.
      • Schaller G.E.
      Ethylene receptors function as components of high-molecular-mass protein complexes in Arabidopsis.
      ). 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 (
      • Hoppen C.
      • Müller L.
      • Hänsch S.
      • Uzun B.
      • Milić D.
      • Meyer A.J.
      • Weidtkamp-Peters S.
      • Groth G.
      Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.
      ). 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 (
      • Dong C.-H.
      • Rivarola M.
      • Resnick J.S.
      • Maggin B.D.
      • Chang C.
      Subcellular co-localization of Arabidopsis RTE1 and ETR1 supports a regulatory role for RTE1 in ETR1 ethylene signaling.
      ,
      • Lin Z.
      • Ho C.W.
      • Grierson D.
      AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis.
      ,
      • Dong C.-H.
      • Jang M.
      • Scharein B.
      • Malach A.
      • Rivarola M.
      • Liesch J.
      • Groth G.
      • Hwang I.
      • Chang C.
      Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1.
      ,
      • Thomine S.
      • Wang R.
      • Ward J.M.
      • Crawford N.M.
      • Schroeder J.I.
      Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes.
      ). A homolog of TRP1 in tomato interacts with both SlETR1 and never ripe (NR or SlETR3) (
      • Lin Z.
      • Arciga-Reyes L.
      • Zhong S.
      • Alexander L.
      • Hackett R.
      • Wilson I.
      • Grierson D.
      SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development.
      ). As will be discussed further below, some of the receptors also interact with components of the cytokinin signaling pathway (
      • Lin Z.
      • Ho C.W.
      • Grierson D.
      AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis.
      ,
      • Dong C.-H.
      • Jang M.
      • Scharein B.
      • Malach A.
      • Rivarola M.
      • Liesch J.
      • Groth G.
      • Hwang I.
      • Chang C.
      Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1.
      ,
      • Scharein B.
      • Voet-van Vormizeele J.
      • Harter K.
      • Groth G.
      Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy.
      ,
      • Urao T.
      • Miyata S.
      • Yamaguchi-Shinozaki K.
      • Shinozaki K.
      Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system.
      ,
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ).
      Two proteins, CTR1 and EIN2, are central components of ethylene signaling (
      • Alonso J.M.
      • Hirayama T.
      • Roman G.
      • Nourizadeh S.
      • Ecker J.R.
      EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis.
      ,
      • Kieber J.J.
      • Rothenberg M.
      • Roman G.
      • Feldmann K.A.
      • Ecker J.R.
      CTR1, A negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases.
      ) that physically interact with the receptors (
      • Zhong S.
      • Lin Z.
      • Grierson D.
      Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum.
      ,
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ,
      • Cancel J.D.
      • Larsen P.B.
      Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis.
      ,
      • Bisson M.M.A.
      • Bleckmann A.
      • Allekotte S.
      • Groth G.
      EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1.
      ,
      • Clark K.L.
      • Larsen P.B.
      • Wang X.
      • Chang C.
      Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors.
      ,
      • Gao Z.
      • Chen Y.F.
      • Randlett M.D.
      • Zhao X.C.
      • Findell J.L.
      • Kieber J.J.
      • Schaller G.E.
      Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes.
      ,
      • Huang Y.F.
      • Li H.
      • Hutchison C.E.
      • Laskey J.
      • Kieber J.J.
      Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis.
      ) and each other (
      • Ju C.
      • Yoon G.M.
      • Shemansky J.M.
      • Lin D.Y.
      • Yin Z.I.
      • Chang J.
      • Garrett W.M.
      • Kessenbrock M.
      • Groth G.
      • Tucker M.L.
      • Cooper B.
      • Kieber J.J.
      • Chang C.
      CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.
      ). CTR1 is a serine/threonine protein kinase that functions as a negative regulator of ethylene signaling (
      • Kieber J.J.
      • Rothenberg M.
      • Roman G.
      • Feldmann K.A.
      • Ecker J.R.
      CTR1, A negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases.
      ). 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 (
      • Alonso J.M.
      • Hirayama T.
      • Roman G.
      • Nourizadeh S.
      • Ecker J.R.
      EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis.
      ). 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 (
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ,
      • Gao Z.
      • Chen Y.F.
      • Randlett M.D.
      • Zhao X.C.
      • Findell J.L.
      • Kieber J.J.
      • Schaller G.E.
      Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes.
      ,
      • Bisson M.M.
      • Groth G.
      New paradigm in ethylene signaling: EIN2, the central regulator of the signaling pathway, interacts directly with the upstream receptors.
      ). These physical interactions appear to be important because mutations in CTR1 that abolish receptor-CTR1 interactions result in a nonfunctional CTR1 (
      • Gao Z.
      • Chen Y.F.
      • Randlett M.D.
      • Zhao X.C.
      • Findell J.L.
      • Kieber J.J.
      • Schaller G.E.
      Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes.
      ,
      • Huang Y.F.
      • Li H.
      • Hutchison C.E.
      • Laskey J.
      • Kieber J.J.
      Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis.
      ), and blocking interactions between ETR1 and EIN2 results in ethylene insensitivity (
      • Bisson M.M.A.
      • Groth G.
      Targeting plant ethylene responses by controlling essential protein-protein interactions in the ethylene pathway.
      ).
      Current models predict that in the absence of ethylene, the ethylene receptors keep CTR1 active (Fig. 3). CTR1 directly phosphorylates EIN2 (
      • Ju C.
      • Yoon G.M.
      • Shemansky J.M.
      • Lin D.Y.
      • Yin Z.I.
      • Chang J.
      • Garrett W.M.
      • Kessenbrock M.
      • Groth G.
      • Tucker M.L.
      • Cooper B.
      • Kieber J.J.
      • Chang C.
      CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.
      ), 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 (
      • Qiao H.
      • Chang K.N.
      • Yazaki J.
      • Ecker J.R.
      Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis.
      ), as hypothesized in several studies (
      • Ju C.
      • Yoon G.M.
      • Shemansky J.M.
      • Lin D.Y.
      • Yin Z.I.
      • Chang J.
      • Garrett W.M.
      • Kessenbrock M.
      • Groth G.
      • Tucker M.L.
      • Cooper B.
      • Kieber J.J.
      • Chang C.
      CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.
      ,
      • Chen R.
      • Binder B.M.
      • Garrett W.M.
      • Tucker M.L.
      • Chang C.
      • Cooper B.
      Proteomic responses in Arabidopsis thaliana seedlings treated with ethylene.
      ,
      • Wen X.
      • Zhang C.
      • Ji Y.
      • Zhao Q.
      • He W.
      • An F.
      • Jiang L.
      • Guo H.
      Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus.
      ,
      • Qiao H.
      • Shen Z.
      • Huang S.-S.C.
      • Schmitz R.J.
      • Urich M.A.
      • Briggs S.P.
      • Ecker J.R.
      Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas.
      ). 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 (
      • Potuschak T.
      • Lechner E.
      • Parmentier Y.
      • Yanagisawa S.
      • Grava S.
      • Koncz C.
      • Genschik P.
      EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins: EBF1 and EBF2.
      ,
      • Guo H.
      • Ecker J.R.
      Plant responses to ethylene gas are mediated by SCF (EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor.
      ,
      • Binder B.M.
      • Walker J.M.
      • Gagne J.M.
      • Emborg T.J.
      • Hemmann G.
      • Bleecker A.B.
      • Vierstra R.D.
      The Arabidopsis EIN3-binding F-box proteins, EBF1 and 2 have distinct but overlapping roles in regulating ethylene signaling.
      ,
      • An F.
      • Zhao Q.
      • Ji Y.
      • Li W.
      • Jiang Z.
      • Yu X.
      • Zhang C.
      • Han Y.
      • He W.
      • Liu Y.
      • Zhang S.
      • Ecker J.R.
      • Guo H.
      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.
      ,
      • Gagne J.M.
      • Smalle J.
      • Gingerich D.J.
      • Walker J.M.
      • Yoo S.D.
      • Yanagisawa S.
      • Vierstra R.D.
      Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation.
      ). 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 thumbnail gr3
      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 (
      • Shakeel S.N.
      • Gao Z.
      • Amir M.
      • Chen Y.-F.
      • Rai M.I.
      • Haq N.U.
      • Schaller G.E.
      Ethylene regulates levels of ethylene receptor/CTR1 signaling complexes in Arabidopsis thaliana.
      ,
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ,
      • Gao Z.
      • Chen Y.F.
      • Randlett M.D.
      • Zhao X.C.
      • Findell J.L.
      • Kieber J.J.
      • Schaller G.E.
      Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes.
      ). 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 (
      • Ju C.
      • Yoon G.M.
      • Shemansky J.M.
      • Lin D.Y.
      • Yin Z.I.
      • Chang J.
      • Garrett W.M.
      • Kessenbrock M.
      • Groth G.
      • Tucker M.L.
      • Cooper B.
      • Kieber J.J.
      • Chang C.
      CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis.
      ,
      • Qiao H.
      • Chang K.N.
      • Yazaki J.
      • Ecker J.R.
      Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis.
      ,
      • Wen X.
      • Zhang C.
      • Ji Y.
      • Zhao Q.
      • He W.
      • An F.
      • Jiang L.
      • Guo H.
      Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus.
      ,
      • Qiao H.
      • Shen Z.
      • Huang S.-S.C.
      • Schmitz R.J.
      • Urich M.A.
      • Briggs S.P.
      • Ecker J.R.
      Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas.
      ).
      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 (
      • Thomine S.
      • Wang R.
      • Ward J.M.
      • Crawford N.M.
      • Schroeder J.I.
      Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes.
      ,
      • Alonso J.M.
      • Hirayama T.
      • Roman G.
      • Nourizadeh S.
      • Ecker J.R.
      EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis.
      ). 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 (
      • Ma B.
      • Zhou Y.
      • Chen H.
      • He S.-J.
      • Huang Y.-H.
      • Zhao H.
      • Lu X.
      • Zhang W.-K.
      • Pang J.-H.
      • Chen S.-Y.
      • Zhang J.-S.
      Membrane protein MHZ3 stabilizes OsEIN2 in rice by interacting with its Nramp-like domain.
      ,
      • Ma B.
      • He S.-J.
      • Duan K.-X.
      • Yin C.-C.
      • Chen H.
      • Yang C.
      • Xiong Q.
      • Song Q.-X.
      • Lu X.
      • Chen H.-W.
      • Zhang W.-K.
      • Lu T.-G.
      • Chen S.-Y.
      • Zhang J.-S.
      Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation.
      ). 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 (
      • Merchante C.
      • Brumos J.
      • Yun J.
      • Hu Q.
      • Spencer K.R.
      • Enríquez P.
      • Binder B.M.
      • Heber S.
      • Stepanova A.N.
      • Alonso J.M.
      Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2.
      ,
      • Li W.
      • Ma M.
      • Feng Y.
      • Li H.
      • Wang Y.
      • Ma Y.
      • Li M.
      • An F.
      • Guo H.
      EIN2-directed translational regulation of ethylene signaling in Arabidopsis.
      ). 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 (
      • Merchante C.
      • Brumos J.
      • Yun J.
      • Hu Q.
      • Spencer K.R.
      • Enríquez P.
      • Binder B.M.
      • Heber S.
      • Stepanova A.N.
      • Alonso J.M.
      Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2.
      ,
      • Li W.
      • Ma M.
      • Feng Y.
      • Li H.
      • Wang Y.
      • Ma Y.
      • Li M.
      • An F.
      • Guo H.
      EIN2-directed translational regulation of ethylene signaling in Arabidopsis.
      ,
      • Olmedo G.
      • Guo H.
      • Gregory B.D.
      • Nourizadeh S.D.
      • Aguilar-Henonin L.
      • Li H.
      • An F.
      • Guzman P.
      • Ecker J.R.
      ETHYLENE-INSENSITIVE5 encodes a 5′ → 3′ exoribonuclease required for regulation of the EIN3-targeting F-box proteins EBF1/2.
      ,
      • Potuschak T.
      • Vansiri A.
      • Binder B.M.
      • Lechner E.
      • Vierstra R.D.
      • Genschik P.
      The exonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis.
      ). 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 (
      • Potuschak T.
      • Lechner E.
      • Parmentier Y.
      • Yanagisawa S.
      • Grava S.
      • Koncz C.
      • Genschik P.
      EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins: EBF1 and EBF2.
      ,
      • Binder B.M.
      • Walker J.M.
      • Gagne J.M.
      • Emborg T.J.
      • Hemmann G.
      • Bleecker A.B.
      • Vierstra R.D.
      The Arabidopsis EIN3-binding F-box proteins, EBF1 and 2 have distinct but overlapping roles in regulating ethylene signaling.
      ,
      • An F.
      • Zhao Q.
      • Ji Y.
      • Li W.
      • Jiang Z.
      • Yu X.
      • Zhang C.
      • Han Y.
      • He W.
      • Liu Y.
      • Zhang S.
      • Ecker J.R.
      • Guo H.
      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 (
      • Zhang F.
      • Wang L.
      • Qi B.
      • Zhao B.
      • Ko E.E.
      • Riggan N.D.
      • Chin K.
      • Qiao H.
      EIN2 mediates direct regulation of histone acetylation in the ethylene response.
      ). 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 (
      • Hildreth S.B.
      • Foley E.E.
      • Muday G.K.
      • Helm R.F.
      • Winkel B.S.J.
      The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is not predicted by changes in the transcriptome.
      ). The exception to this model is that short-term, transient responses occur independently of these transcription factors yet require EIN2 (
      • Binder B.M.
      • Mortimore L.A.
      • Stepanova A.N.
      • Ecker J.R.
      • Bleecker A.B.
      Short-term growth responses to ethylene in Arabidopsis seedlings are EIN3/EIL1 independent.
      ). 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 (
      • Alonso J.M.
      • Stepanova A.N.
      • Solano R.
      • Wisman E.
      • Ferrari S.
      • Ausubel F.M.
      • Ecker J.R.
      Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis.
      ,
      • Chao Q.
      • Rothenberg M.
      • Solano R.
      • Roman G.
      • Terzaghi W.
      • Ecker J.R.
      Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins.
      ,
      • Solano R.
      • Stepanova A.
      • Chao Q.
      • Ecker J.R.
      Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1.
      ). 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 (
      • Poulios S.
      • Vlachonasios K.E.
      Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.
      ,
      • Li C.
      • Xu J.
      • Li J.
      • Li Q.
      • Yang H.
      Involvement of Arabidopsis histone acetyltransferase HAC family genes in the ethylene signaling pathway.
      ,
      • Zhou C.
      • Zhang L.
      • Duan J.
      • Miki B.
      • Wu K.
      HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.
      ). Additionally, research has identified specific histone acetylation marks that are important in ethylene-regulated gene expression by EIN3 (
      • Wang F.
      • Wang L.
      • Qiao L.
      • Chen J.
      • Pappa M.B.
      • Pei H.
      • Zhang T.
      • Chang C.
      • Dong C.H.
      Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.
      ,
      • Zhang F.
      • Qi B.
      • Wang L.
      • Zhao B.
      • Rode S.
      • Riggan N.D.
      • Ecker J.R.
      • Qiao H.
      EIN2-dependent regulation of acetylation of histone H3K14 and non-canonical histone H3K23 in ethylene signalling.
      ,
      • Zhang F.
      • Wang L.
      • Ko E.E.
      • Shao K.
      • Qiao H.
      Histone deacetylases SRT1 and SRT2 interact with ENAP1 to mediate ethylene-induced transcriptional repression.
      ). 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 (
      • Hildreth S.B.
      • Foley E.E.
      • Muday G.K.
      • Helm R.F.
      • Winkel B.S.J.
      The dynamic response of the Arabidopsis root metabolome to auxin and ethylene is 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 (
      • Binder B.M.
      • Mortimore L.A.
      • Stepanova A.N.
      • Ecker J.R.
      • Bleecker A.B.
      Short-term growth responses to ethylene in Arabidopsis seedlings are EIN3/EIL1 independent.
      ,
      • Kim J.
      • Wilson R.L.
      • Case J.B.
      • Binder B.
      A comparative study of ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery.
      ,
      • Rai M.I.
      • Wang X.
      • Thibault D.M.
      • Kim H.J.
      • Bombyk M.M.
      • Binder B.M.
      • Shakeel S.N.
      • Schaller G.E.
      The ARGOS gene family functions in a negative feedback loop to desensitize plants to ethylene.
      ,
      • Prescott A.M.
      • McCollough F.W.
      • Eldreth B.L.
      • Binder B.M.
      • Abel S.M.
      Analysis of network topologies underlying ethylene growth response kinetics.
      ,
      • Shi J.
      • Drummond B.J.
      • Wang H.
      • Archibald R.L.
      • Habben J.E.
      Maize and Arabidopsis ARGOS proteins interact with ethylene receptor signaling complex, supporting a regulatory role for ARGOS in ethylene signal transduction.
      ,
      • Deslauriers S.D.
      • Alvarez A.A.
      • Lacey R.F.
      • Binder B.M.
      • Larsen P.B.
      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.
      ,
      • Konishi M.
      • Yanagisawa S.
      Ethylene signaling in Arabidopsis involves feedback regulation via the elaborate control of EBF2 expression by EIN3.
      ,
      • Shi J.
      • Habben J.E.
      • Archibald R.L.
      • Drummond B.J.
      • Chamberlin M.A.
      • Williams R.W.
      • Lafitte H.R.
      • Weers B.P.
      Overexpression of ARGOS genes modifies plant sensitivity to ethylene, leading to improved drought tolerance in both Arabidopsis and maize.
      ,
      • Robles L.M.
      • Wampole J.S.
      • Christians M.J.
      • Larsen P.B.
      Arabidopsis enhanced ethylene response 4 encodes an EIN3-interacting TFIID transcription factor required for proper ethylene response, including ERF1 induction.
      ,
      • Christians M.J.
      • Larsen P.B.
      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.
      ,
      • Christians M.J.
      • Robles L.M.
      • Zeller S.M.
      • Larsen P.B.
      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.
      ,
      • Kim H.G.
      • Kwon S.J.
      • Jang Y.J.
      • Nam M.H.
      • Chung J.H.
      • Na Y.-C.
      • Guo H.
      • Park O.K.
      GDSL LIPASE1 modulates plant immunity through feedback regulation of ethylene signaling.
      ,
      • Kim H.G.
      • Kwon S.J.
      • Jang Y.J.
      • Chung J.H.
      • Nam M.H.
      • Park O.K.
      GDSL lipase 1 regulates ethylene signaling and ethylene-associated systemic immunity in Arabidopsis.
      ,
      • Kwon S.J.
      • Jin H.C.
      • Lee S.
      • Nam M.H.
      • Chung J.H.
      • Kwon S.I.
      • Ryu C.-M.
      • Park O.K.
      GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling 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 (
      • Qu X.
      • Schaller G.E.
      Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.
      ,
      • Wang W.
      • Hall A.E.
      • O'Malley R.
      • Bleecker A.B.
      Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission.
      ,
      • Cancel J.D.
      • Larsen P.B.
      Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis.
      ,
      • Hua J.
      • Meyerowitz E.M.
      Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana.
      ,
      • Ciardi J.A.
      • Tieman D.M.
      • Lund S.T.
      • Jones J.B.
      • Stall R.E.
      • Klee H.J.
      Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression.
      ,
      • Tieman D.M.
      • Taylor M.G.
      • Ciardi J.A.
      • Klee H.J.
      The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family.
      ,
      • Hall A.E.
      • Bleecker A.B.
      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) (
      • Rai M.I.
      • Wang X.
      • Thibault D.M.
      • Kim H.J.
      • Bombyk M.M.
      • Binder B.M.
      • Shakeel S.N.
      • Schaller G.E.
      The ARGOS gene family functions in a negative feedback loop to desensitize plants to ethylene.
      ,
      • Shi J.
      • Habben J.E.
      • Archibald R.L.
      • Drummond B.J.
      • Chamberlin M.A.
      • Williams R.W.
      • Lafitte H.R.
      • Weers B.P.
      Overexpression of ARGOS genes modifies plant sensitivity to ethylene, leading to improved drought tolerance in both Arabidopsis and maize.
      ,
      • Resnick J.S.
      • Wen C.-K.
      • Shockey J.A.
      • Chang C.
      REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis.
      ,
      • Barry C.S.
      • Giovannoni J.J.
      Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling.
      ). An RTE1-like protein, green ripe (GR), has a similar role in tomato (
      • Barry C.S.
      • Giovannoni J.J.
      Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling.
      ). The exact mechanisms for regulation by these proteins are under investigation. More information about this is contained in a recent review (
      • Azhar B.J.
      • Zulfiqar A.
      • Shakeel S.N.
      • Schaller G.E.
      Amplification and adaptation in the ethylene signaling pathway.
      ).
      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 (
      • Gamble R.L.
      • Coonfield M.L.
      • Schaller G.E.
      Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis.
      ,
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Zutz T.C.
      • Bleecker A.B.
      Ethylene stimulates nutations that are dependent on the ETR1 receptor.
      ,
      • Wilson R.L.
      • Bakshi A.
      • Binder B.M.
      Loss of the ETR1 ethylene receptor reduces the inhibitory effect of far-red light and darkness on seed germination of Arabidopsis thaliana.
      ,
      • Wilson R.L.
      • Kim H.
      • Bakshi A.
      • Binder B.M.
      The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress.
      ,
      • Bakshi A.
      • Piya S.
      • Fernandez J.C.
      • Chervin C.
      • Hewezi T.
      • Binder B.M.
      Ethylene receptors signal via a non-canonical pathway to regulate abscisic acid responses.
      ,
      • Bakshi A.
      • Wilson R.L.
      • Lacey R.F.
      • Kim H.
      • Wuppalapati S.K.
      • Binder B.M.
      Identification of regions in the receiver domain of the ETHYLENE RESPONSE1 ethylene receptor of Arabidopsis important for functional divergence.
      ,
      • Desikan R.
      • Hancock J.T.
      • Bright J.
      • Harrison J.
      • Weir I.
      • Hooley R.
      • Neill S.J.
      A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells.
      ,
      • Qiu L.
      • Xie F.
      • Yu J.
      • Wen C.-K.
      Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1.
      ,
      • Kim J.
      • Patterson S.E.
      • Binder B.M.
      Reducing jasmonic acid levels causes ein2 mutants to become ethylene responsive.
      ). 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 (
      • Bakshi A.
      • Piya S.
      • Fernandez J.C.
      • Chervin C.
      • Hewezi T.
      • Binder B.M.
      Ethylene receptors signal via a non-canonical pathway to regulate abscisic acid responses.
      ). 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 (
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Moore J.M.
      • Parks B.M.
      • Spalding E.P.
      • Bleecker A.B.
      Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis.
      ,
      • Wilson R.L.
      • Kim H.
      • Bakshi A.
      • Binder B.M.
      The ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE2 have contrasting roles in seed germination of Arabidopsis during salt stress.
      ,
      • Bakshi A.
      • Piya S.
      • Fernandez J.C.
      • Chervin C.
      • Hewezi T.
      • Binder B.M.
      Ethylene receptors signal via a non-canonical pathway to regulate abscisic acid responses.
      ,
      • Qu X.
      • Schaller G.E.
      Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.
      ,
      • Wang W.
      • Hall A.E.
      • O'Malley R.
      • Bleecker A.B.
      Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission.
      ,
      • Hall B.P.
      • Shakeel S.N.
      • Amir M.
      • Ul Haq N.
      • Qu X.
      • Schaller G.E.
      Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis.
      ,
      • Street I.H.
      • Aman S.
      • Zubo Y.
      • Ramzan A.
      • Wang X.
      • Shakeel S.N.
      • Kieber J.J.
      • Schaller G.E.
      Ethylene inhibits cell proliferation of the Arabidopsis root meristem.
      ,
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ,
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ). 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 (
      • Schaller G.E.
      • Shiu S.-H.
      • Armitage J.P.
      Two-component systems and their co-option for eukaryotic signal transduction.
      ,
      • Kieber J.J.
      • Schaller G.E.
      Cytokinin signaling in plant development.
      ). 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 (
      • Scharein B.
      • Voet-van Vormizeele J.
      • Harter K.
      • Groth G.
      Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy.
      ,
      • Urao T.
      • Miyata S.
      • Yamaguchi-Shinozaki K.
      • Shinozaki K.
      Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system.
      ,
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ,
      • Scharein B.
      • Groth G.
      Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1.
      ). This interaction involves the C-terminal portion of ETR1 (
      • Scharein B.
      • Voet-van Vormizeele J.
      • Harter K.
      • Groth G.
      Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy.
      ,
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ). 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 (
      • Scharein B.
      • Groth G.
      Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1.
      ).
      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 (
      • Binder B.M.
      • Kim H.J.
      • Mathews D.E.
      • Hutchison C.E.
      • Kieber J.J.
      • Schaller G.E.
      A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene.
      ,
      • Street I.H.
      • Aman S.
      • Zubo Y.
      • Ramzan A.
      • Wang X.
      • Shakeel S.N.
      • Kieber J.J.
      • Schaller G.E.
      Ethylene inhibits cell proliferation of the Arabidopsis root meristem.
      ,
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ,
      • Mira-Rodado V.
      • Veerabagu M.
      • Witthöft J.
      • Teply J.
      • Harter K.
      • Desikan R.
      Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana.
      ). Null mutants of ARR1 are less responsive to ethylene, and this appears to depend upon ETR1 histidine kinase activity (
      • Street I.H.
      • Aman S.
      • Zubo Y.
      • Ramzan A.
      • Wang X.
      • Shakeel S.N.
      • Kieber J.J.
      • Schaller G.E.
      Ethylene inhibits cell proliferation of the Arabidopsis root 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 (
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Moore J.M.
      • Parks B.M.
      • Spalding E.P.
      • Bleecker A.B.
      Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis.
      ,
      • Binder B.M.
      • Kim H.J.
      • Mathews D.E.
      • Hutchison C.E.
      • Kieber J.J.
      • Schaller G.E.
      A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene.
      ). Additionally, ETR1 histidine kinase activity is involved in both ethylene- and cytokinin-induced changes in root apical meristem (
      • Zdarska M.
      • Cuyacot A.R.
      • Tarr P.T.
      • Yamoune A.
      • Szmitkowska A.
      • Hrdinová V.
      • Gelová Z.
      • Meyerowitz E.M.
      • Hejátko J.
      ETR1 integrates response to ethylene and cytokinins into a single multistep phosphorelay pathway to control root growth.
      ). 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 (
      • Nemhauser J.L.
      • Hong F.
      • Chory J.
      Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses.
      ), 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 (
      • Zhao H.
      • Duan K.-X.
      • Ma B.
      • Yin C.-C.
      • Hu Y.
      • Tao J.-J.
      • Huang Y.-H.
      • Cao W.-Q.
      • Chen H.
      • Yang C.
      • Zhang Z.-G.
      • He S.-J.
      • Zhang W.-K.
      • Wan X.-Y.
      • Lu T.-G.
      • et al.
      Histidine kinase MHZ1/OsHK1 interacts with ethylene receptors to regulate root growth in rice.
      ). 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 (
      • Voet-van-Vormizeele J.
      • Groth G.
      Ethylene controls autophosphorylation of the histidine kinase domain in ethylene receptor ETR1.
      ,
      • Bisson M.M.A.
      • Groth G.
      New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2.
      ), whereas genetic experiments suggest that ethylene leads to more phosphotransfer (
      • Binder B.M.
      • O'Malley R.C.
      • Wang W.
      • Moore J.M.
      • Parks B.M.
      • Spalding E.P.
      • Bleecker A.B.
      Arabidopsis seedling growth response and recovery to ethylene: a kinetic analysis.
      ,
      • Qu X.
      • Schaller G.E.
      Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1.
      ). Histidine kinases can carry out multiple enzymatic reactions, including kinase, phosphatase, and phosphotransfer reactions, and receiver domains can catalyze both phosphotransfer and autodephosphorylation reactions (
      • Gao R.
      • Stock A.M.
      Biological insights from structures of two-component proteins.
      ,
      • Bourret R.B.
      Receiver domain structure and function in response regulator proteins.
      ). 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 occur until ethylene binds to the receptor to bring the DHp (site of histidine phosphorylation) and receiver domains into the correct orientation. Thus, ethylene may be increasing phosphotransfer through the pathway, causing the steady-state level of ETR1 histidine phosphorylation to decrease. This will only be answered conclusively when we have structural data.
      Noncanonical signaling is also likely to occur downstream of the receptors. For instance, PpCTR1 in Physcomitrella patens has a role in both ethylene and ABA signal transduction, raising the possibility that CTR1 has more functions than simply phosphorylating EIN2 (
      • Yasumura Y.
      • Pierik R.
      • Kelly S.
      • Sakuta M.
      • Voesenek L.A.C.J.
      • Harberd N.P.
      An ancestral role for CONSTITUTIVE TRIPLE RESPONSE1 proteins in both ethylene and abscisic acid signaling.
      ). Also, mutants of EIN2 have altered responses to various hormones (reviewed in Ref.
      • Van de Poel B.
      • Smet D.
      • Van Der Straeten D.
      Ethylene and hormonal cross talk in vegetative growth and development.
      ), but whether this reflects alternative signaling from EIN2 or is due to many pathways converging on EIN2 has yet to be completely explored.
      The signaling pathway downstream of EIN2 is complex because it involves at least two levels of transcriptional regulation. Because of this, it is harder to distinguish “canonical” from “noncanonical” signaling. EIN3 is the transcription factor with the largest role in ethylene signaling (
      • Binder B.M.
      • Walker J.M.
      • Gagne J.M.
      • Emborg T.J.
      • Hemmann G.
      • Bleecker A.B.
      • Vierstra R.D.
      The Arabidopsis EIN3-binding F-box proteins, EBF1 and 2 have distinct but overlapping roles in regulating ethylene signaling.
      ,
      • Alonso J.M.
      • Stepanova A.N.
      • Solano R.
      • Wisman E.
      • Ferrari S.
      • Ausubel F.M.
      • Ecker J.R.
      Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis.
      ), and it homodimerizes to interact with its target DNA (
      • Solano R.
      • Stepanova A.
      • Chao Q.
      • Ecker J.R.
      Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1.
      ). However, environmental factors such as dark versus light or the presence of other hormones can affect this so that, depending on conditions, EIN3 interacts with other transcription factors, leading to outputs not predicted by the common ethylene-signaling models (
      • Zhu Z.
      • An F.
      • Feng Y.
      • Li P.
      • Xue L.
      • Jiang A.M.Z.
      • Kim J.-M.
      • To T.K.
      • Li W.
      • Zhang X.
      • Yu Q.
      • Dong Z.
      • Chen W.-Q.
      • Seki M.
      • Zhou J.-M.
      • Guo H.
      Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis.
      ,
      • He X.
      • Jiang J.
      • Wang C.-Q.
      • Dehesh K.
      ORA59 and EIN3 interaction couples jasmonate-ethylene synergistic action to antagonistic salicylic acid regulation of PDF expression.