Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis

Ethylene is an important regulator of plant growth, development and responses to environmental stresses. Arabidopsis perceives ethylene through five homologous receptors that negatively regulate ethylene responses. RTE1, a novel gene conserved in plants, animals and some protists, was recently identified as a positive regulator of the ETR1 ethylene receptor. Here, we genetically analyze the dependence of ETR1 on RTE1 in order to obtain further insight into RTE1 function. The function of RTE1 was found to be independent and distinct from that of RAN1, which encodes a copper transporter required for ethylene receptor function. We tested the ability of an rte1 loss-of-function mutation to suppress 11 etr1 ethylene-binding domain mis-sense mutations, all of which result in dominant ethylene insensitivity due to constitutive signaling. This suppression test uncovered two classes of etr1 mutations -RTE1-dependent and RTE1-independent. The nature of these mutations suggests that the ethylene-binding domain is a possible target of RTE1 action. Based on these findings, we propose that RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain.     

The Relationship between Ethylene Binding and Dominant Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene Receptor

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.     

Mutational analysis of the ethylene receptor ETR1. Role of the histidine kinase domain in dominant ethylene insensitivity

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The N-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The C-terminal half of the polypeptide contains domains with homology to histidine (His) kinases and response regulators, signaling motifs originally identified in bacteria. The role of the His kinase domain in ethylene signaling was examined in planta. For this purpose, site-directed mutations were introduced into the full-length wild-type ETR1 gene and into etr1-1, a mutant allele that confers dominant ethylene insensitivity on plants. The mutant forms of the receptor were expressed in Arabidopsis and the transgenic plants characterized for their ethylene responses. A mutation that eliminated His kinase activity did not affect the ability of etr1-1 to confer ethylene insensitivity. A truncated version of etr1-1 that lacks the His kinase domain also conferred ethylene insensitivity. Possible mechanisms by which a truncated version of etr1-1 could exert dominance are discussed.     

Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis

Ethylene plays important roles in plant growth, development and stress responses, and is perceived by a family of receptors that repress ethylene responses when ethylene is absent. Repression by the ethylene receptor ETR1 depends on an integral membrane protein, REVERSION TO ETHYLENE SENSITIVITY1 (RTE1), which acts upstream of ETR1 in the endoplasmic reticulum (ER) membrane and Golgi apparatus. To investigate RTE1 function, we screened for RTE1‐interacting proteins using the yeast split‐ubiquitin assay, which yielded the ER‐localized cytochrome b₅ (Cb5) isoform D. Cb5s are small hemoproteins that perform electron transfer reactions in all eukaryotes, but their roles in plants are relatively uncharacterized. Using bimolecular fluorescence complementation (BiFC), we found that all four ER‐localized Arabidopsis Cb5 isoforms (AtCb5–B, ‐C, ‐D and ‐E) interact with RTE1 in plant cells. In support of this interaction, atcb5 mutants exhibited phenotypic parallels with rte1 mutants in Arabidopsis. Phenotypes included partial suppression of etr1–2 ethylene insensitivity, and no suppression of RTE1‐independent ethylene receptor isoforms. The single loss‐of‐function mutants atcb5–b, ‐c and ‐d appeared similar to the wild‐type, but double mutant combinations displayed slight ethylene hypersensitivity. Over‐expression of AtCb5–D conferred reduced ethylene sensitivity similar to that conferred by RTE1 over‐expression, and genetic analyses suggested that AtCb5–D acts upstream of RTE1 in the ethylene response. These findings suggest an unexpected role for Cb5, in which Cb5 and RTE1 are functional partners in promoting ETR1‐mediated repression of ethylene signaling.     

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed.     

Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr1^1-349 or the dominant ethylene-insensitive etr1-1^1-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr1^1-349 and etr1-1^1-349, with etr1-1^1-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr1^1-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr1^1-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR1^7-560 prevented ethylene receptor signaling, and the CTR1^7-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr1^1-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.The gaseous plant hormone ethylene is perceived by a small family of ethylene receptors. Arabidopsis (Arabidopsis thaliana) has five ethylene receptors that are structurally similar to prokaryotic two-component histidine kinase (HK) proteins. Mutants defective in multiple ethylene receptor genes show a constitutive ethylene response phenotype, which indicates a negative regulation of ethylene responses by the receptor genes (Hua and Meyerowitz, 1998).The receptor N terminus has three or four transmembrane domains that bind ethylene. The GAF (for cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which follows the transmembrane helices, mediates noncovalent receptor heterodimerization and may have a role in receptor cooperation (Gamble et al., 2002; O’Malley et al., 2005; Xie et al., 2006; Gao et al., 2008). The subfamily I receptors Ethylene Response1 (ETR1) and Ethylene Response Sensor1 (ERS1) have a conserved HK domain following the GAF domain. For subfamily II members ETR2, Ethylene Insensitive4 (EIN4), and ERS2, the HK domain is less conserved, and they lack most signature motifs essential for HK activity (Chang et al., 1993; Gamble et al., 1998; Hua et al., 1998; Qu and Schaller, 2004; Xie et al., 2006). Among the five receptors, ETR1, ETR2, and EIN4 have a receiver domain following the HK domain. The ETR1 HK domain may have a role in mediating the receptor signal to downstream components, and the HK activity facilitates the ethylene signaling (Clark et al., 1998; Huang et al., 2003; Hall et al., 2012). The receiver domain can dimerize and could involve receptor cooperation (Müller-Dieckmann et al., 1999). However, differential receptor cooperation occurs between the receiver domain-lacking ERS1 and the other ethylene receptors, which does not support the hypothesis that the domains involve receptor cooperation (Liu and Wen, 2012).Acting downstream of the ethylene receptors is Constitutive Triple Response1 (CTR1), a MEK kinase (mitogen-activated protein kinase kinase kinase) with Ser/Thr kinase activity, and the kinase domain locates at the C terminus. The CTR1 N terminus does not share sequence similarity to known domains and can physically interact with the ethylene-receptor HK domain (Clark et al., 1998; Huang et al., 2003). ctr1 mutants showing attenuated CTR1 kinase activity or the ETR1-CTR1 association exhibit various degrees of the constitutive ethylene-response phenotype. For example, the ctr1-1 and ctr1^btk mutations result from the D694E and E626K substitutions, respectively, in the CTR1 kinase domain, and ctr1-1 shows a stronger ethylene-response phenotype than ctr1^btk, with ctr1-1 having much weaker kinase activity than ctr1^btk (Kieber et al., 1993; Huang et al., 2003; Ikeda et al., 2009). The ctr1-8 mutation results in the G354E substitution that prevents the ETR1-CTR1 association, and the mutant exhibits a constitutive ethylene-response phenotype. Overexpression of the CTR1 N terminus CTR1^7-560, which is responsible for interaction with ethylene receptors, leads to constitutive ethylene responses, possibly by titrating out available ethylene receptors (Kieber et al., 1993; Huang et al., 2003). These studies suggest that CTR1 kinase activity and the interaction of CTR1 with the receptor HK domain may be important to the ethylene receptor signal output in suppressing constitutive ethylene responses.Although the ETR1-CTR1 interaction via the HK domain is essential to the ethylene receptor signal output, evidence suggests that the ETR1 receptor signal output can also be independent of the HK activity or domain. The etr1 ers1 loss-of-function mutant displays extreme growth defects. The etr1[HGG] mutation inactivates ETR1 HK activity, and expression of the getr1[HGG] transgene rescues the etr1 ers1 growth defects, which indicates a lack of association of ETR1 receptor signaling and its kinase activity (Wang et al., 2003). The dominant etr1-1 mutation results in the C65Y substitution and confers ethylene insensitivity (Chang et al., 1993), and the expression of the HK domain-lacking etr1^1-349 and ethylene-insensitive etr1-1^1-349 isoforms partially suppresses the growth defects of etr1 ers1-2. Loss-of-function mutations of subfamily II members do not affect etr1-1^1-349 functions. Therefore, etr1-1^1-349 predominantly cooperates with subfamily I receptors to mediate the ethylene receptor signal output (Xie et al., 2006). Biochemical and transformation studies showing that ethylene receptors can form heterodimers and that each receptor is a component of high-molecular-mass complexes explain how ethylene receptors may act cooperatively (Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010).Reversion To Ethylene Sensitivity1 (RTE1), a Golgi/endoplasmic reticulum protein, was isolated from a suppressor screen of the dominant ethylene-insensitive etr1-2 mutation. The cross-species complementation of the rte1-2 loss-of-function mutation by the rice (Oryza sativa) RTE Homolog1 (OsRTH1) suggests a conserved mechanism that modulates the ethylene receptor signaling across higher plant species (Zhang et al., 2012). RTE1 and OsRTH1 overexpression led to ethylene insensitivity in wild-type Arabidopsis but not the etr1-7 loss-of-function mutant, and expression of etr1^1-349 restored ethylene insensitivity with RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007; Zhang et al., 2010). Coimmunoprecipitation of epitope-tagged ETR1 and RTE1 and Trp fluorescence spectroscopy revealed the physical interaction of RTE1 and ETR1 (Zhou et al., 2007; Dong et al., 2008, 2010). Therefore, RTE1 may directly promote ETR1 receptor signal output through the ETR1 N terminus, but whether RTE1 has an essential role in ETR1 N-terminal signaling remains to be addressed.Currently, the biochemical nature of the ethylene receptor signal is unknown, and the underlying mechanisms of mediation of the ethylene receptor signal output remain uninvestigated. Genetic and biochemical studies suggest that activation of CTR1 by ethylene receptors may suppress constitutive ethylene responses; upon ethylene binding, the receptors are converted to an inactive state and fail to activate CTR1, and the suppression of ethylene responses by CTR1 is alleviated (Hua and Meyerowitz, 1998; Klee, 2004; Wang et al., 2006; Hall et al., 2007). However, this model does not address how the ETR1 N terminus, which does not have the CTR1-interacting site, mediates the receptor signal to suppress constitutive ethylene responses. The receptor signal of the truncated etr1 isoforms may be mediated by other full-length ethylene receptors and then activate CTR1; alternatively, the ETR1 N-terminal signal may be mediated by a pathway independent of CTR1 (Gamble et al., 2002; Qu and Schaller, 2004; Xie et al., 2006). Results showing that mutants defective in multiple ethylene receptor genes exhibit a more severe ethylene-response phenotype than ctr1 and that ctr1 mutants are responsive to ethylene support the presence of a CTR1-independent pathway (Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Huang et al., 2003; Liu et al., 2010).In this study, we investigated whether mediation of ETR1 N-terminal signaling is independent of CTR1 and whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling. The ETR1 N-terminal signaling was not mediated via other full-length ethylene receptors, but the signal of full-length ethylene receptors could be mediated by the ETR1 N terminus independent of CTR1. The ETR1 C terminus may inhibit ETR1 N-terminal signaling, whereby deletion of the C terminus facilitates N-terminal signaling. We propose a model for the possible modulation of ETR1 receptor signaling.     

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

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

Possible modulation of Arabidopsis ETR1 N-terminal signaling by CTR1

The mitogen-activated protein kinase kinase kinase (MAPKKK) Constitutive Triple-Response1 (CTR1) plays a key role in mediating ethylene receptor signaling via its N-terminal interaction with the ethylene receptor C-terminal histidine kinase (HK) domain. Loss-of-function mutations of CTR1 prevent ethylene receptor signaling, and corresponding ctr1 mutants show a constitutive ethylene response phenotype. We recently reported in Plant Physiology that expression of the truncated ethylene receptor Ethylene Response1 (ETR1) isoforms etr11-349 and dominant ethylene-insensitive etr1-11-349, lacking the C-terminal HK and receiver domains, both suppressed the ctr1 mutant phenotype. Therefore, the ETR1 N terminus is capable of receptor signaling independent of CTR1. The constitutive ethylene response phenotype is stronger for ctr1-1 than ctr1-1 lines expressing the etr11-349 transgene, so N-terminal signaling by the full-length but not truncated ETR1 is inhibited by ctr1-1. We address possible modulations of ETR1 N-terminal signaling with docking of CTR1 on the ETR1 HK domain.     

Ethylene stimulates nutations that are dependent on the ETR1 receptor

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.     

Arabidopsis ETR1 and ERS1 differentially repress the ethylene response in combination with other ethylene receptor genes

The ethylene response is negatively regulated by a family of five ethylene receptor genes in Arabidopsis (Arabidopsis thaliana). The five members of the ethylene receptor family can physically interact and form complexes, which implies that cooperativity for signaling may exist among the receptors. The ethylene receptor gene mutations etr1-1((C65Y))(for ethylene response1-1), ers1-1((I62P)) (for ethylene response sensor1-1), and ers1(C65Y) are dominant, and each confers ethylene insensitivity. In this study, the repression of the ethylene response by these dominant mutant receptor genes was examined in receptor-defective mutants to investigate the functional significance of receptor cooperativity in ethylene signaling. We showed that etr1-1((C65Y)), but not ers1-1((I62P)), substantially repressed various ethylene responses independent of other receptor genes. In contrast, wild-type receptor genes differentially supported the repression of ethylene responses by ers1-1((I62P)); ETR1 and ETHYLENE INSENSITIVE4 (EIN4) supported ers1-1((I62P)) functions to a greater extent than did ERS2, ETR2, and ERS1. The lack of both ETR1 and EIN4 almost abolished the repression of ethylene responses by ers1(C65Y), which implied that ETR1 and EIN4 have synergistic effects on ers1(C65Y) functions. Our data indicated that a dominant ethylene-insensitive receptor differentially repressed ethylene responses when coupled with a wild-type ethylene receptor, which supported the hypothesis that the formation of a variety of receptor complexes may facilitate differential receptor signal output, by which ethylene responses can be repressed to different extents. We hypothesize that plants can respond to a broad ethylene concentration range and exhibit tissue-specific ethylene responsiveness with differential cooperation of the multiple ethylene receptors.     

Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), ethylene is perceived by a receptor family consisting of five members. Subfamily 1 members ETHYLENE RESPONSE1 (ETR1) and ETHYLENE RESPONSE SENSOR1 (ERS1) have histidine kinase activity, unlike the subfamily 2 members ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4), which lack amino acid residues critical for this enzymatic activity. To resolve the role of histidine kinase activity in signaling by the receptors, we transformed an etr1-9;ers1-3 double mutant with wild-type and kinase-inactive versions of the receptor ETR1. Both wild-type and kinase-inactive ETR1 rescue the constitutive ethylene-response phenotype of etr1-9;ers1-3, restoring normal growth to the mutant in air. However, the lines carrying kinase-inactive ETR1 exhibit reduced sensitivity to ethylene based on several growth response assays. Microarray and real-time polymerase chain reaction analyses of gene expression support a role for histidine kinase activity in eliciting the ethylene response. In addition, protein levels of the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which physically associates with the ethylene receptor ETR1, are less responsive to ethylene in lines containing kinase-inactive ETR1. These data indicate that the histidine kinase activity of ETR1 is not required for but plays a modulating role in the regulation of ethylene responses. Models for how enzymatic and nonenzymatic regulation may facilitate signaling from the ethylene receptors are discussed.     

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

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

Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1

In Arabidopsis, ethylene is perceived by a receptor family consisting of five members, one of these being ETR1. The N-terminal half of ETR1 functions as a signal input domain. The C-terminal region of ETR1, consisting of a His kinase domain and a putative receiver domain, is likely to function in signal output. The role of the proposed signal output region in ethylene signaling was examined in planta. For this purpose, the ability of mutant versions of ETR1 to rescue the constitutive ethylene-response phenotype of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant line was examined. A truncated version of ETR1 that lacks both the His kinase domain and the receiver domain failed to rescue the triple mutant phenotype. A truncated ETR1 receptor that lacks only the receiver domain restored normal growth to the triple mutant in air, but the transgenic seedlings displayed hypersensitivity to low doses of ethylene. A mutation that eliminated His kinase activity had a modest effect upon the ability of the receptor to repress ethylene responses in air. These results demonstrate that the His kinase domain plays a role in the repression of ethylene responses. The potential roles of the receiver domain and His kinase activity in ethylene signaling are discussed.     

The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination

In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.     

Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.     

Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Background  

Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.     

Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis

Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response.