Ethylene Sensitivity and Response Sensor Expression in Petioles of Rumex Species at Low O2 and High CO2 Concentrations

Rumex palustris, a flooding-tolerant plant, elongates its petioles in response to complete submergence. This response can be partly mimicked by enhanced ethylene levels and low O2 concentrations. High levels of CO2 do not markedly affect petiole elongation in R. palustris. Experiments with ethylene synthesis and action inhibitors demonstrate that treatment with low O2 concentrations enhances petiole extension by shifting sensitivity to ethylene without changing the rate of ethylene production. The expression level of the R. palustris gene coding for the putative ethylene receptor (RP-ERS1) is up-regulated by 3% O2 and increases after 20 min of exposure to a low concentration of O2, thus preceding the first significant increase in elongation observable after 40 to 50 min. In the flooding-sensitive species Rumex acetosa, submergence results in a different response pattern: petiole growth of the submerged plants is the same as for control plants. Exposure of R. acetosa to enhanced ethylene levels strongly inhibits petiole growth. This inhibitory effect of ethylene on R. acetosa can be reduced by both low levels of O2 and/or high concentrations of CO2.     

Ethylene Biosynthesis and Accumulation under Drained and Submerged Conditions (A Comparative Study of Two Rumex Species)

A model is presented of the regulation of ethylene biosynthesis in relation to submergence and flooding resistance. It is based on time-course measurements of ethylene production, ethylene accumulation, and concentrations of free and conjugated 1-aminocyclo-propane-1-carboxylic acid (ACC) in submerged and drained flooding-resistant Rumex palustris Sm. and flooding-sensitive Rumex acetosella L. plants. From these data, in vivo reaction rates of the final steps in the ethylene biosynthetic pathway were calculated. According to our model, submergence stimulates ACC formation and inhibits conversion of ACC to ethylene in both Rumex species, and as a result, ACC accumulates. This may explain the stimulated ACC conjugation observed in submerged plants. Although submergence inhibited ethylene production, physical entrapment increased endogenous ethylene concentrations in both flooding-resistant R. palustris and flooding-sensitive R. acetosella plants. However, R. palustris plants controlled their internal ethylene levels in the long term by a negative regulation of ACC synthase induced by ethylene. In flooding-sensitive R. acetosella plants, absence of negative regulation increased internal ethylene levels to more than 20 [mu]L L-1 after 6 d of submergence. This may accelerate the process of senescence and contribute to their low level of flooding resistance.     

Heteromeric interactions among ethylene receptors mediate signaling in Arabidopsis

The gaseous hormone ethylene is perceived in Arabidopsis by a five member receptor family that consists of the subfamily 1 receptors ETR1 and ERS1 and the subfamily 2 receptors ETR2, ERS2, and EIN4. Previous work has demonstrated that the basic functional unit for the ethylene receptor, ETR1, is a disulfide-linked homodimer. We demonstrate here that ethylene receptors isolated from Arabidopsis also interact with each other through noncovalent interactions. Evidence that ETR1 associates with other ethylene receptors was obtained by co-purification of ETR1 with tagged versions of ERS1, ETR2, ERS2, and EIN4 from Arabidopsis membrane extracts. ETR1 preferentially associated with the subfamily 2 receptors compared with the subfamily 1 receptor ERS1, but ethylene treatment affected the interactions and relative composition of the receptor complexes. When transgenically expressed in yeast, ETR1 and ERS2 can form disulfide-linked heterodimers. In plant extracts, however, the association of ETR1 and ERS2 can be largely disrupted by treatment with SDS, supporting a higher order noncovalent interaction between the receptors. Yeast two-hybrid analysis demonstrated that the receptor GAF domains are capable of mediating heteromeric receptor interactions. Kinetic analysis of ethylene-insensitive mutants of ETR1 is consistent with their dominance being due in part to an ability to associate with other ethylene receptors. These data suggest that the ethylene receptors exist in plants as clusters in a manner potentially analogous to that found with the histidine kinase-linked chemoreceptors of bacteria and that interactions among receptors contribute to ethylene signal output.     

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.     

Ethylene perception by the ERS1 protein in Arabidopsis

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.     

Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments

A cDNA from deep water rice treated with ethylene, encoding an ethylene receptor homologous to Arabidopsis thaliana ETR2 and EIN4, was isolated using differential display and RACE techniques. The cDNA (2880 bp), corresponding to the Os-ERL1 gene (Oryza sativa ETHYLENE RESPONSE 2 like 1; GenBank accession number AB107219), contained an open reading frame of 2289 bp coding for 763 amino acids. The protein Os-ERL1 has 50% and 52% similarity to Arabidopsis ETR2 and EIN4, respectively. The Os-ERL1 gene was up-regulated by flooding, and by treatment with ethylene and gibberellin. These results suggest that deep water rice responds to flooding by increasing the number of its ethylene receptors.     

ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana

Ethylene influences many processes in Arabidopsis thaliana through the action of five receptor isoforms. All five isoforms use copper as a cofactor for binding ethylene. Previous research showed that silver can substitute for copper as a cofactor for ethylene binding activity in the ETR1 ethylene receptor yet also inhibit ethylene responses in plants. End-point and rapid kinetic analyses of dark-grown seedling growth revealed that the effects of silver are mostly dependent upon ETR1, and ETR1 alone is sufficient for the effects of silver. Ethylene responses in etr1-6 etr2-3 ein4-4 triple mutants were not blocked by silver. Transformation of these triple mutants with cDNA for each receptor isoform under the promoter control of ETR1 revealed that the cETR1 transgene completely rescued responses to silver while the cETR2 transgene failed to rescue these responses. The other three isoforms partially rescued responses to silver. Ethylene binding assays on the binding domains of the five receptor isoforms expressed in yeast showed that silver supports ethylene binding to ETR1 and ERS1 but not the other isoforms. Thus, silver may have an effect on ethylene signaling outside of the ethylene binding pocket of the receptors. Ethylene binding to ETR1 with silver was ～30% of binding with copper. However, alterations in the K(d) for ethylene binding to ETR1 and the half-time of ethylene dissociation from ETR1 do not underlie this lower binding. Thus, it is likely that the lower ethylene binding activity of ETR1 with silver is due to fewer ethylene binding sites generated with silver versus copper.     

Ethylene Receptors Function as Components of High-Molecular-Mass Protein Complexes in Arabidopsis

Background

The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4.

Methodology/Principal Findings

Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding.

Conclusions/Significance

These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.     

Submergence-Induced Ethylene Synthesis,Entrapment, and Growth in Two Plant Species with Contrasting Flooding Resistances

Submergence-induced ethylene synthesis and entrapment were studied in two contrasting Rumex species, one flood-resistant (Rumex palustris) and the other flood-sensitive (Rumex acetosa). The application of a photoacoustic method to determine internal ethylene concentrations in submerged plants is discussed. A comparison with an older technique (vacuum extraction) is described. For the first time ethylene production before, during, and after submergence and the endogenous concentration during submergence were continuously measured on a single intact plant without physical perturbation. Both Rumex species were characterized by enhanced ethylene concentrations in the shoot after 24 h of submergence. This was not related to enhanced synthesis but to continued production and physical entrapment. In R. palustris, high endogenous ethylene levels correlated with enhanced petiole and lamina elongation. No dramatic change in leaf growth rate was observed in submerged R. acetosa shoots. After desubmergence both species showed an increase in ethylene production, the response being more pronounced in R. palustris. This increase was linked to the enhanced postsubmergence growth rate of leaves of R. palustris. Due to the very rapid escape of ethylene out of desubmerged plants to the atmosphere (90% disappeared within 1 min), substantial underestimation of internal ethylene concentrations can be expected using more conventional vacuum extraction techniques.     

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.     

Characterization of an ethylene receptor homologue from wheat and its expression during leaf senescence

A wheat ethylene receptor homologue (W-er1) was isolated from a wheat stem cDNA library using the Arabidopsis ETR1 cDNA as a probe. The predicted amino acid sequence of W-er1 is over 70% similar to ERS1 from Arabidopsis and exhibits homology to bacterial two-component response regulators within the histidine kinase domain. Northern hybridization demonstrated that W-er1 was expressed in stem, leaf and root tissues. Treatments known to induce senescence of detached leaves including jasmonate, abscisic acid and wounding, increased the accumulation of W-er1 mRNA, while benzyladenine treatment did not. These data suggest that W-er1 may play a role in the process of leaf senescence.     

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors <Emphasis Type="Italic">ETR1</Emphasis> and <Emphasis Type="Italic">ERS1</Emphasis>

Background  

The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations.     

An Ethylene-Mediated Increase in Sensitivity to Auxin Induces Adventitious Root Formation in Flooded Rumex palustris Sm

The hormonal regulation of adventitious root formation induced by flooding of the root system was investigated in the wetland species Rumex palustris Sm. Adventitious root development at the base of the shoot is an important adaptation to flooded conditions and takes place soon after the onset of flooding. Decreases in either endogenous auxin or ethylene concentrations induced by application of inhibitors of either auxin transport or ethylene biosynthesis reduced the number of adventitious roots formed by flooded plants, suggesting an involvement of these hormones in the rooting process. The rooting response during flooding was preceded by increased endogenous ethylene concentrations in the root system. The endogenous auxin concentration did not change during flooding-induced rooting, but a continuous basipetal transport of auxin from the shoot to the rooting zone appeared to be essential in maintaining stable auxin concentrations. These results suggest that the higher ethylene concentration in soil-flooded plants increases the sensitivity of the root-forming tissues to endogenous indoleacetic acid, thus initiating the formation of adventitious roots.     

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii

We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 and ERS1 and designated them Cm-ETR1 (C. melo ETR1; accession no. AF054806) and Cm-ERS1 (C. melo ERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1 mRNA in the pericarp increased in parallel with the increase in fruit size and then markedly decreased at the end of enlargement. In fully enlarged fruit the level of Cm-ERS1 mRNA was low in all tissues, whereas that of Cm-ETR1 mRNA was very high in the seeds and placenta. During ripening Cm-ERS1 mRNA increased slightly in the pericarp of fruit before the marked increase of Cm-ETR1 mRNA paralleled climacteric ethylene production. These results indicate that both Cm-ETR1 and Cm-ERS1 play specific roles not only in ripening but also in the early development of melon fruit and that they have distinct roles in particular fruit tissues at particular developmental stages.     

Contrasting interactions between ethylene and abscisic acid in Rumex species differing in submergence tolerance

Complete submergence of flooding-tolerant Rumex palustris plants strongly stimulates petiole elongation. This escape response is initiated by the accumulation of ethylene inside the submerged tissue. In contrast, petioles of flooding-intolerant Rumex acetosa do not increase their elongation rate under water even though ethylene also accumulates when they are submerged. Abscisic acid (ABA) was found to be a negative regulator of enhanced petiole growth in both species. In R. palustris, accumulated ethylene stimulated elongation by inhibiting biosynthesis of ABA via a reduction of RpNCED expression and enhancing degradation of ABA to phaseic acid. Externally applied ABA inhibited petiole elongation and prevented the upregulation of gibberellin A(1) normally found in submerged R. palustris. In R. acetosa submergence did not stimulate petiole elongation nor did it depress levels of ABA. However, if ABA concentrations in R. acetosa were first artificially reduced, submergence (but not ethylene) was then able to enhance petiole elongation strongly. This result suggests that in Rumex a decrease in ABA is a prerequisite for ethylene and other stimuli to promote elongation.