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EIN4 and ERS2 Are Members of the Putative Ethylene Receptor Gene Family in ArabidopsisJian Huaa, Hajime Sakaia, Saeid Nourizadehb, Qianhong G. Chenc, Anthony B. Bleeckerc, Joseph R. Eckerb, and Elliot M. Meyerowitzaa Division of Biology, 156-29, California Institute of Technology, Pasadena, California 91125 b Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 c Botany Department, Birge Hall, University of Wisconsin, Madison, Wisconsin 53706 Correspondence to: Elliot M. Meyerowitz, meyerow{at}cco.caltech.edu (E-mail), 626-449-0756 (fax).
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.
The simple gas ethylene acts as an endogenous regulator of growth and development as well as a mediator of stress responses in higher plants. It is involved in many developmental processes, including seed germination, leaf and flower senescence, and fruit ripening (
Recently, progress has been made in understanding the signal transduction pathway of ethylene. This has been achieved largely by molecular and genetic studies that use the model plant Arabidopsis. More than a dozen ethylene response mutants have been identified by screening for alterations in the triple response (
The emerging picture is that ethylene signal transduction is performed by a phosphorylation cascade that might modulate the activity of transcription factors regulating gene expression. The ethylene signal appears to be perceived by a family of ethylene receptors. Three members of the putative ethylene receptor gene family, ETR1, ERS (for ETHYLENE RESPONSE SENSOR), and ETR2, have been cloned ( We have gained insight into ethylene perception by identifying the ethylene receptor gene ETR1 and the two putative ethylene receptor genes ETR2 and ERS. However, additional components may be involved in this process, as suggested by the following observations. First, the genetic screens for ethylene response mutants are not saturated, as indicated by the availability of only one allele at some loci and by the failure to find ers mutants. Second, mutants with similar ethylene-insensitive phenotypes have been identified, but their molecular characteristics have not been determined. One such mutant is ein4, which had a dominant ethylene-insensitive phenotype in the triple response assay. Like the members in the putative ethylene receptor gene family, EIN4 acts upstream of CTR1. Therefore, we attempted to isolate more members of the putative ethylene receptor family in Arabidopsis through sequence homology. Here, we describe the cloning of two ETR2 homologous genes and show that one of them is the EIN4 gene. The other, ERS2, is also involved in ethylene perception, as indicated by transgenic mutant analysis. We analyzed the RNA expression patterns of members of this gene family and the regulation of their expression levels by ethylene. The structure and function of this gene family are discussed.
Isolation of Two Homologs of the ETR2 Gene
Identification of One Homolog as the EIN4 Gene We isolated two new ein4 alleles (designated as ein4-2 and ein4-3) from screens for ethylene-insensitive mutants. They both exhibited dominant ethylene-insensitive phenotypes in the triple response assay (Figure 1A). ein4-2 was obtained from diepoxybutane mutagenesis. When crossed to ein4-1, it failed to segregate any wild-type seedlings in 679 F2 progeny. ein4-3 was isolated from an ethylmethane sulfate–mutagenized population. Cosegregation of ein4-3 and ein4-1 was not tested, but this mutant is likely to be an ein4 allele, because it maps close to EIN4 (between nga172 and nga162). We identified a missense mutation in each ein4 allele (see below). On the basis of its map position, its sequence homology to the putative ethylene receptor genes, and the mutations in this gene in all three ein4 alleles, we concluded that this homolog is the EIN4 gene.
Approximately 3200 bp of the genomic DNA of EIN4 and its longest cDNA (~2990 bp) were sequenced. The size of the longest cDNA was approximately the same as the size of the corresponding RNA transcript on an RNA gel blot (data not shown), indicating that this cDNA is full length, or nearly so. Comparing the genomic DNA and cDNA sequences revealed that EIN4 contains one intron. The predicted protein has 766 amino acids with a predicted molecular mass of 86 kD. It has an overall 53% identity and 74% similarity to ETR2 in protein sequences and 62 and 60% similarity to ETR1 and ERS, respectively. That EIN4 is more related to ETR2 than to ETR1 or ERS was also evident when individual domains were compared. The N-terminal domain (positions 1 to 347) of EIN4 has 61% sequence identity and 78% similarity to that of ETR2. Like ETR2, it has an extra hydrophobic stretch of amino acids at the very N terminus when aligned to ETR1 and ERS (Figure 2A). The middle portion of the EIN4 protein (positions 348 to 631) is a putative histidine protein kinase domain. Similar to the corresponding domain of ETR2, it is more diverged than those of ETR1 and ERS from the canonical sequences of the bacterial protein histidine kinases (Figure 2B). It is 69% similar to the corresponding portion of ETR2 and 53% similar to those of ETR1 and ERS. However, unlike ETR2, the putative autophosphorylation site histidine residue (H-377) is present at the predicted site. The C-terminal domain has sequence similarity to the receiver domains of ETR2 (70% similarity) and ETR1 (66% similarity; Figure 2C). The two aspartates (D-648 and D-694) and a lysine residue (K-746) conserved in the bacterial two-component regulators are present at the predicted positions in EIN4.
We sequenced the EIN4 gene from the three ein4 mutants; in each allele, we observed a missense mutation (Figure 2A). Both ein4-1 and ein4-2 contained an A-to-T transversion, which results in a substitution of isoleucine at position 84 by phenylalanine in the putative transmembrane segment II. This change is the same as those in etr1-4 and ers-1 (
ERS2 Is a New Member of the Putative Ethylene Receptor Family The predicted ERS2 protein is related more closely to ETR2 and EIN4 (73 and 68% similarity) than it is to ETR1 or ERS1 (58 and 57% similarity). The N-terminal domain (positions 1 to 362) has 81 and 75% similarity to those of ETR2 and EIN4, respectively. Like ETR2 and EIN4, it has a fourth putative transmembrane segment at the N-terminal end (Figure 2A). The three hydrophobic segments (I, II, and III), which are essential for ethylene binding in ETR1, are conserved in ERS2. The C-terminal domain of ERS2 exhibits 63, 58, 53, and 47% similarity to the putative histidine kinase domains of ETR2, EIN4, ETR1, and ERS1, respectively. It is the most divergent from the canonical form of the bacterial histidine protein kinases among the five proteins. Most of the signature motifs of histidine kinases are not easily recognizable in ERS2, and it lacks the histidine residue at the predicted autophosphorylation site. Also, unlike the other four proteins, ERS2 contains a unique stretch of six serines and five glutamic acids close to the end of the protein (Figure 2B). The ERS2 gene was mapped to the top of chromosome 1 by RFLP mapping. The newly released sequences from the Arabidopsis genome project contain the gene F19P19.21, which appears to be the same as ERS2. The F19P19.21 annotated sequence has a 16–amino acid deletion from C-470 to R-485 and has an extra six amino acids between K-592 and Y-593 of the predicted ERS2 sequence. These discrepancies apparently are due to annotation errors. No previously identified mutations reside close to the chromosomal region where the ERS2 gene maps. To identify its function, we used a reverse genetics approach. Its sequence homology to the ethylene receptor gene ETR1 suggested that ERS2 might also play a role in ethylene perception. The functions of other members of the gene family were suggested by their dominant mutant phenotypes resulting from missense mutations in their N-terminal domains. Therefore, we engineered similar missense mutations into the ERS2 gene and introduced the mutated genes into wild-type Arabidopsis plants.
We isolated a 6-kb EcoRI genomic fragment of the ERS2 gene containing the coding region, 4 kb of 5' upstream sequence, and 230 bp of 3' sequence after the translation stop codon. Because the 3' fragment of the cloned ERS2 gene might not have been sufficient for RNA processing, a 1-kb 3' end of the nopaline synthase (NOS) gene was added. Two mutant ERS2 genes were generated by in vitro mutagenesis. The mutant ERS2 P67L gene has proline substituted by leucine at position 67, which is a mutation similar to that of etr2-1 ( The ethylene-insensitive phenotype of ers2-1 and ers2-2 extends to responses other than the triple response. Leaf expansion of the mutant plants was not inhibited by the exogenous application of ethylene (data not shown). Moreover, mutant plants had larger leaves than did the wild type when grown in air, which might be due to their insensitivity to endogenous ethylene. Double mutant analysis was performed to place ERS2 in the ethylene signal transduction pathway. ctr1 mutants have the constitutive ethylene response phenotype. Etiolated ctr1 seedlings show the triple response when grown in air, and the adult ctr1 plants have stunted growth as if they were constantly being treated with ethylene. Double mutants between ers2 and ctr1 mimicked the ctr1 phenotype. Etiolated seedlings of the double mutants had constitutive triple response when grown in air (Figure 1C). The adult plants basically resembled the ctr1 single mutants, although they had slightly more expanded leaves than did ctr1. From these results, we determined that ERS2 acts upstream of CTR1 and thus occupies a position in the ethylene signal transduction pathway similar to other putative ethylene receptors. Thus, ERS2 defines a new member of the putative ethylene receptor gene family.
Expression Patterns of the Ethylene Receptor–Related Gene Family To further analyze the expression of each member of this gene family at the cellular level, we performed RNA in situ hybridization with a number of tissues. The RNA levels of the five genes were generally low and ubiquitous, and they had basically similar expression patterns in most of the tissues (Figure 3 and Figure 4).
ERS1 was ubiquitously expressed. In general, the signals appeared higher in younger and smaller cells than they did in older and more expanded cells. It remains to be determined whether the number of RNA molecules expressed per cell is the same in both types of cells. ERS1 was expressed in embryos (Figure 3A and Figure 3B), leaves, and etiolated seedlings, including cotyledons (Figure 3C and Figure 3D), hypocotyls (Figure 3E and Figure 3F), and roots (Figure 3G and Figure 3H). ERS1 expression was observed in stems, especially in procambium cells (Figure 3I and Figure 3J). Young floral primordia and floral organ primordia expressed ERS strongly (Figure 3K and Figure 3L). Very strong expression was observed in the locules of the anthers (Figure 3M to P). ERS1 also was expressed in the developing septum of the carpels (Figure 3Q and Figure 3R) and was later expressed in carpels, including the ovules. ETR1 had an expression pattern similar to ERS1 in etiolated seedlings and leaves but appeared to be expressed at a lower level. Expression of ETR1 in stems and flowers was of similar pattern and comparable level to that of ERS1. Like ERS1, ETR1 was strongly expressed in the locules of the anthers (Figure 4A and Figure 4B) and the developing carpels (Figure 4C and Figure 4D). It is also expressed in the ovules and funiculi in late carpel development (Figure 4E and Figure 4F).
The ETR2 expression pattern was similar to that of ETR1 and ERS1 in the embryos, etiolated seedlings, leaves, stems, and young floral primordia, but the flower expression pattern was slightly different. Strong expression was not observed in the stamens (Figure 4G and Figure 4H), in contrast to ETR1 (Figure 4A and Figure 4B) and ERS1 (Figure 3M and Figure 3N). ETR2 was strongly expressed in the developing carpels, especially in the funiculi and ovules (Figure 4I and Figure 4J; EIN4 was expressed in embryos, etiolated seedlings, leaves, roots, and inflorescences. Strong signals were detected in the locules of stamens, including the developing pollen cells and tapetum cells (Figure 4K and Figure 4L). Developing carpels expressed EIN4 at moderate levels (data not shown). ERS2 expression was detected in the etiolated seedlings, leaves, roots, and stems. Higher expression was observed in flowers. Stamens, especially the tapetum cells and developing pollen cells, as well as developing carpels, ovules, and funiculi expressed ERS2 strongly (Figure 4M to P). Later in flower development, ERS2 expression was concentrated in the epidermal layers of the septum (Figure 4Q and Figure 4R), where strong expression was not observed with the other four genes.
Because these five genes are involved in ethylene signaling, we analyzed the regulation of their RNA expression by ethylene. It has been noted that ETR1 is not regulated by ethylene treatment (
Three members of the putative ethylene receptor gene family have been found in Arabidopsis; they are ETR1, ERS1, and ETR2 ( The five genes appear to belong to two subfamilies (Figure 6). One subfamily consists of ETR1 and ERS1, whereas the other contains ETR2, EIN4, and ERS2. Members in the same subfamily have higher amino acid sequence similarity to each other than to members in the other subfamily. The five proteins share 79% and 68 to 74% similarity within each subfamily, respectively, and 57 to 65% similarity between subfamilies. Moreover, genes in the same subfamily have conserved intron positions that are not shared between the two subfamilies (Figure 6). ETR1 has five introns that correspond in length and position to introns in ERS1, with the exception of the sixth intron in the receiver domain, which ERS1 does not contain. ETR2, EIN4, and ERS2, on the other hand, exhibit one intron at a location where no introns are found in either ETR1 or ERS1. This division of subfamilies is not related to the presence or absence of the receiver domain, because the two members without the receiver domain (ERS1 and ERS2) belong to different subfamilies.
The most conserved domain of these proteins is the N-terminal domain, with similarities ranging from 64 to 82% among members. The three hydrophobic segments at the N terminus exhibit even higher sequence conservation (Figure 2A). These putative transmembrane segments may potentially form the ethylene binding pocket, because the first 165 amino acids of ETR1, which is made up mostly of these segments, are capable of ethylene binding in yeast (
The putative histidine kinase domains of this gene family differ greatly in their sequence similarities to the bacterial histidine kinases (Figure 2B). ETR1 and ERS1 have substantial homology to the bacterial proteins in this region. All of the five signature motifs, H, N, G1, F, and G2 ( Despite the similarity and divergence in sequences, all of these five genes when mutated conferred similar dominant mutant phenotypes. Mutant plants are insensitive to ethylene in a variety of responses, including triple response, leaf growth, and leaf senescence. There does not seem to be any tissue specificity associated with mutants of different genes, except for the variation in strengths. Our in situ hybridization data are consistent with the pleiotropic effects of these mutations: the genes are expressed ubiquitously, and they have largely overlapping expression domains. It is desirable to have loss-of-function mutations to analyze the functions of EIN4 and ERS2. Thus far, only dominant mutations of the two genes are available, like the other three members of the gene family. Depending on the mechanisms of the dominance of these mutations, several scenarios regarding their functions can be envisioned. It remains possible that EIN4 and ERS2 may not encode ethylene receptors. The dominant ein4 and ers2 mutations may be neomorphic, that is, the mutant EIN4 and ERS2 proteins may exhibit a function normally not associated with their wild-type activities. The mutant proteins could interfere with the function of the ethylene receptor, such as ETR1, because they exhibit sequence homology to the real receptor. Alternately, EIN4 and ERS2 may encode ethylene receptors, and the five genes in the family may perform similar functions. This functional redundancy could explain the failure to isolate recessive alleles of any of the genes in the genetic screens. In this scenario, the mutations induce constitutive activity of the proteins or cause dominant interference of the wild-type activities of other receptors. Total redundancy raises the question of how all of these genes could be kept from becoming pseudogenes during evolution. It appears more likely that these genes are somewhat different, while sharing common functions in ethylene sensing. Differences in expression are indeed observed among family members. These genes are differentially regulated by ethylene: the RNA levels of the ERS1, ETR2, and ERS2 genes are upregulated by ethylene, whereas the expression of ETR1 and EIN4 is not affected by ethylene treatment. In addition, the spatial expression patterns of these genes are overlapping but not identical. For instance, ERS2 has a unique expression domain in the epidermal layer of the septum, and ETR2 expression is below detection in the stamen locules, where the other four members show strong expression.
Ethylene response has been shown to be regulated at the level of ethylene synthesis. But this regulation does not account for all of the regulation in ethylene responses. It has been observed in several plant species that ethylene sensitivity varies widely between tissues, between classes of response within a tissue, and even within a single class of response under different physiological conditions (
It is conceivable that these genes may not act independently if they all encode ethylene receptors. Heterodimer or heteromultimer forms of these receptors could be used for ethylene sensing. They could even act in several linear pathways, with some members acting on the others. Multiple-step phosphorelay systems consisting of H-to-D-to-H-to-D have been discovered in two-component systems, such as Kin A-Spo0 and BvgS-BvgA in bacteria and Sln1p-Ypd1p-Ssk1p in yeast ( More in-depth studies on ethylene signal transduction are now made possible by the identification of this putative ethylene receptor gene family and by the availability of mutants with altered response due to changes in each member. Isolation and characterization of loss-of-function alleles of these genes should reveal the functions of the wild-type proteins and further our understanding of the process of ethylene perception.
Plant Growth and Genetic Studies
ein4-3 was crossed to ecotype Landsberg erecta for mapping. F3 families homozygous for the ethylene-insensitive phenotype were used for simple sequence length polymorphism mapping (
Library Screening and DNA Analysis To identify mutations in the ein4 alleles, we amplified the EIN4 coding regions from wild-type and mutant plant tissues by using polymerase chain reaction (PCR). PCR products were excised from low-melting-point gels and used directly for sequencing. The alterations in sequence were verified by independent PCR amplifications.
Restriction fragment length polymorphism (RFLP) mapping was done with the MAPMAKER program (
In Vitro Mutagenesis and Plant Transformation
RNA in Situ Hybridization and RNA Isolation
We thank Leonard Medrano for his assistance in RFLP mapping and Alfred Wang for his assistance in RNA in situ hybridization experiments. We thank Xuemei Chen, Jennifer Fletcher, Toshiro Ito, Steve Jackson, Prakash Kumar, Carolyn Ohno, Kevin Roberg, Doris Wagner, and Eva Ziegelhoffer for critical reading of the manuscript. This work was supported by U.S. Department of Energy Grant No. 88ER13873 to E.M.M. Received March 3, 1998; accepted June 22, 1998.
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clones that hybridized with ETR2 at reduced stringency but did not hybridize with ETR1, ERS, and ETR2 at high stringency were chosen for further analysis. Ten positive clones were isolated. Cross-hybridization between these clones indicated that they belong to two groups. Sequence analysis revealed that each group represented an ETR2-related gene. cDNA clones of these two genes subsequently were isolated from a cDNA library. 
CTA and ATT
