BACKGROUND
ethylene is a gaseous hormone regulating many aspects of plant growth and development.
the dicotyledonous model plant arabidopsis has five ethylene receptors that physically act at the endoplasmic reticulum  with the raf-like protein constitutive triple-response <dig>  to negatively regulate ethylene signaling  <cit> .
in the absence of ethylene, the receptor signal output is mediated by an unknown mechanism to activate ctr <dig>  and ctr <dig> is presumably activated to phosphorylate the downstream signaling component ethylene insensitive <dig> .
phosphorylated ein <dig> stays at the er and cannot induce the ethylene response.
with ethylene binding to ethylene receptors, the receptor signal output is prevented and ctr <dig> is not activated.
unphosphorylated ein <dig> undergoes proteolytic cleavage by an unknown mechanism to produce a c-terminal fragment, which enters the nucleus to induce the ethylene response  <cit> .
targets of the ein <dig> c terminus remain to be identified.
the transcription factors ein <dig> and ein3-like <dig>  direct the expression of ethylene-responsive genes  <cit> , and ein <dig> c-terminus–induced ethylene responses are prevented in the ein3- <dig> loss-of-function mutant  <cit> .
conceivably, the ein <dig> c terminus could mediate the ethylene signaling to ein <dig> and eil <dig>
the ethylene receptors are structurally similar to histidine kinases  of prokaryotic two-component modules, and studies have revealed the domain functions of the ethylene receptors.
the n terminus has three or four transmembrane domains  that bind a copper cofactor for ethylene binding and are required for localization at the er  <cit> .
following the tms is the gaf domain for non-covalent receptor interaction to mediate inter-receptor signaling  <cit> .
the c-terminal portion is the hk domain, which is believed to function in the receptor signal output via direct interaction with the ctr <dig> n terminus  <cit> .
ctr <dig> has serine/threonine kinase activity, and the ethylene response is inversely associated with ctr <dig> kinase activity  <cit> .
these studies suggest that the hk domain mediates ethylene receptor signaling to the ctr <dig> n terminus, thus activating ctr <dig> to suppress the ethylene response, although the underlying biochemical mechanisms are elusive.
recent studies suggest that the hk domain can be dispensable to ethylene receptor signal output and that ctr <dig> is not the only component mediating the signaling.
mutations that delete ethylene response <dig>  hk and receiver domains have little effect on the receptor signal output  <cit> , and expression of the truncated etr11- <dig> isoform that lacks the site for interacting with ctr <dig> rescues the ctr <dig> loss-of-function mutant phenotype.
conceivably, the etr <dig> receptor signal output can be mediated via the n terminus to an alternative pathway independent of ctr <dig>  <cit> .
of note, etr <dig> receptor signaling in kinase-defective ctr <dig> mutants occurs only when hk-domain–lacking etr <dig> isoforms are expressed.
a kinase-defective ctr <dig> isoform may dock at the etr <dig> hk domain and actively prevent etr <dig> n-terminal signaling to the ctr1-independent alternative pathway  <cit> .
the ctr1- <dig> mutation results from a t-dna insertion at the 5′-untranslated region  of ctr <dig> <cit> .
the 5′-utr of a mrna may be highly structured and contain upstream augs  and internal ribosome entry sites that can affect the translation.
with increased upstream open reading frames  and a highly structured nature, a long 5′-utr can be translationally inhibitory  <cit> .
with a higher ctr <dig> level in ctr1- <dig> than in the wild type  <cit> , the t-dna insertion may substantially increase the 5′-utr length of the ctr <dig> transcript to affect translation efficiency, thus reducing the ctr <dig> level and thus activity as compared with the wild type.
the underlying biochemical mechanisms of ctr <dig> activation by the ethylene receptors remain to be unraveled.
mutations that enhance the constitutive ethylene response in ctr1- <dig> could have a role involving ctr <dig> activation, protein stability or ethylene signaling.
here we report that the loss-of-function mutation of enhancing ctr1- <dig> ethylene response <dig>  enhanced the ethylene response in ctr1- <dig> comparable to the strong allele ctr1- <dig> and the ethylene-treated wild type.
results from extensive genetic and transformation studies suggested that ecr <dig> acts downstream of the ethylene receptors and upstream of the positive ethylene response regulators ein <dig> and eil <dig>  we discuss possible roles for ecr <dig> in the negative regulation of the ethylene response.
RESULTS
ctr1- <dig> is a weak allele
the ctr1- <dig> mutation results from a t-dna insertion at the 5′-utr, and the mutant shows a weak constitutive ethylene response phenotype  <cit> .
the nature of the mutation that causes the weak phenotype remains to be determined.
given that the t-dna inserts at the 5′-utr, we expected that the t-dna fragment is transcribed as part of the 5′-utr of ctr1- <dig> mrna .
indeed, rt-pcr with primers pairing the 5′-utr and t-dna sequences showed the presence of a chimeric cdna fragment containing the sequence of the ctr1- <dig> 5′-utr and the t-dna .
for the same gene, a longer 5′-utr can be translationally more inhibitory than a shorter one  <cit> .
with a  <dig> -kb t-dna at the 5′-utr, we hypothesized that ctr1- <dig> could produce a lower level of ctr <dig> than the wild type, for a weak constitutive ethylene response phenotype.
ethylene inhibits hypocotyl growth and the ethylene response can be quantified by measurement of hypocotyls.
hypocotyls were shorter for ctr1- <dig> than wild-type  seedlings over a wide range of ethylene concentrations .
when growth for wild-type and ctr1- <dig> seedlings are normalized to growth with no added ethylene, it becomes apparent that ctr1- <dig> seedlings are more sensitive to ethylene with a shift from  <dig> μl l- <dig> for wild-type to  <dig>  μl l- <dig> for the mutant .
these data suggest increased ethylene sensitivity with the ctr1- <dig> mutation.
genetic screen for ctr1- <dig> enhancer mutations
to isolate components that could be involved in ctr1-mediated ethylene receptor signaling, we performed an enhancer screen for ctr1- <dig>  alleles that enhanced the ctr1- <dig> mutant phenotype were designated enhancing ctr1- <dig> ethylene response .
here we describe the isolation of ecr2- <dig> ctr1- <dig>
without ethylene treatment, etiolated seedlings of ecr2- <dig> ctr1- <dig> and the strong allele ctr1- <dig> were identical in phenotype and hypocotyl length ; ctr1- <dig> seedlings were longer than the two genotypes and shorter than the wild type  .
as expected, ethylene treatment inhibited the seedling growth in the wild type , ctr1- <dig>  and ecr2- <dig> ctr1- <dig> .
of note, ecr2- <dig> ctr1- <dig> seedlings already showed a constitutive ethylene response phenotype, with a short hypocotyl, so that the effect of ethylene on the seedling growth inhibition was minor .
the silver ion ag may bind etr <dig> and prevent ethylene inhibition of etr <dig> receptor signaling  <cit> .
silver treatment largely prevented ethylene-induced inhibition of seedling hypocotyl growth in wild-type  and ctr1- <dig> but not ecr2- <dig> ctr1- <dig> seedlings .
of note, the seedling hypocotyls were longer for silver-treated than non-silver–treated ecr2- <dig> ctr1- <dig> seedlings , regardless of ethylene treatment; the difference was minor and could be of little biological significance.
these results suggest that etr <dig> receptor signaling that was mediated in part in ctr1- <dig> was prevented in ecr2- <dig> ctr1- <dig>
aminoethoxyvinylglycine  prevents the biosynthesis of the immediate ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic , and the treatment can reduce endogenous ethylene production  <cit> .
avg  treatment did not rescue the reduced growth of ecr2- <dig> ctr1- <dig> seedlings, so ecr2- <dig> did not enhance the production of ethylene to cause reduced growth .
ethylene overproducer <dig>  is a negative regulator of the acc-synthesing enzyme acc synthase <dig>  and the eto1- <dig> allele produces a higher level of acc, and therefore ethylene, than does the wild type  <cit> .
as a control, eto1- <dig> seedlings showed a short hypocotyl, and growth was rescued with avg treatment ; avg treatment in this study was sufficient to prevent endogenous ethylene production.
we examined the growth inhibition phenotype at other developmental stages.
grown under light, ethylene treatment inhibited cotyledon expansion and root elongation in wild-type  seedlings.
seedlings of ctr1- <dig>  ctr1- <dig>  and ecr2- <dig> ctr1- <dig> showed the growth inhibition phenotype regardless of ethylene treatment .
at the adult stage, rosettes were larger for the wild type  than the three mutants, with ctr1- <dig> and ecr2- <dig> ctr1- <dig> being phenotypically similar and both producing a smaller rosette than ctr1- <dig> .
we examined alterations in other aspects of the ethylene response.
leaves of the wild type, ctr1- <dig>  ctr1- <dig>  and ecr2- <dig> ctr1- <dig> showed the senescence phenotype to various degrees 5 days after detachment .
detached leaves of the mutants showed a similar chlorophyll a level, with a similar or slightly higher level in ctr1- <dig> than ctr1- <dig> and ecr2- <dig> ctr1- <dig>  .
ethylene response factor <dig>  expression is directed by ein <dig> and the expression is associated with degrees of ethylene response  <cit> .
erf <dig> expression was slightly higher in ctr1- <dig> than the wild type and highly induced in ctr1- <dig> and ecr2- <dig> ctr1- <dig> without ethylene treatment .
ctr <dig> levels in ctr1- <dig>  ctr1- <dig>  and ecr2- <dig> ctr1- <dig> were identical  and higher than in the wild type .
the ecr2- <dig> allele did not affect ctr <dig> expression in ctr1- <dig>
ecr2- <dig> is a recessive, loss-of-function mutation
to genetically evaluate whether the effect of the ecr2- <dig> mutation on the ctr1- <dig> mutant phenotype is associated with single or multiple alleles, we crossed ecr2- <dig> ctr1- <dig> with the wild type  and ctr1- <dig>  in the f <dig>  generation of the wild type  cross,  <dig> individuals were scored, and only  <dig> showed the ecr2- <dig> ctr1- <dig> growth inhibition phenotype .
in the f <dig> generation of the ctr1- <dig> cross,  <dig> individuals were scored, and  <dig> showed the ecr2- <dig> ctr1- <dig> growth inhibition phenotype .
results from both genetic analyses suggested that the ecr2- <dig> loss-of-function mutation was recessive and intergenic to ctr1- <dig>  the independent segregation of ecr2- <dig> and ctr1- <dig> suggested that ecr <dig> and ctr <dig> are in distinct linkage groups.
we attempted to clone ecr <dig> by map-based cloning and mapped ecr <dig> to a 598-kb region spanning the centromere at chromosome  <dig> , which agreed with ecr <dig> and ctr <dig>  being in distinct linkage groups.
however, we did not obtain more recombinants to advance the mapping to a narrower region.
this region contains sequence gaps  and fragments with repeated sequences, retrotransposon, transposase, and transposable elements.
we sequenced  <dig> annotated genes  but did not identify any mutation .
among the genes, t-dna insertion mutants for at2g07981and at2g <dig> were each genetically crossed with ctr1- <dig> to test whether any of these was ecr <dig>  and these mutations did not enhance the ctr1- <dig> phenotype.
the mutation that affects ecr <dig> remained to be identified and we attempted to isolate the ecr2- <dig> single mutant.
the f <dig> seedlings from the genetic crossing of ecr2- <dig> ctr1- <dig> and the wild type  were crossed with ctr1- <dig>  and the individual that produced the ecr2- <dig> ctr1- <dig> mutant phenotype in the f <dig> generation with a 1: <dig> segregation ratio was considered the ecr2- <dig> mutant.
we isolated ecr2- <dig>  and results from  <dig> independent crossings with ctr1- <dig> showed a 1: <dig> segregation .
grown in dark, ecr2- <dig> seedlings produced a shorter hypocotyl than did the wild type  at low ethylene concentrations ; without ethylene treatment, the height of ecr2- <dig> seedlings was  <dig> ± <dig>  mm  shorter than that of wild-type seedlings.
when this data was normalized, we found that wild-type and ecr2- <dig> seedlings had indistinguishable sensitivity to ethylene .
although the relative hypocotyl length was longer  for ecr2- <dig> seedlings than wild-type seedlings with  <dig> μl l- <dig> ethylene, the difference was small and could be of little biological significance.
at the adult stage, the rosette was smaller for ecr2- <dig> than the wild type .
we excluded the possibility that the mutant phenotype of ecr2- <dig> and ecr2- <dig> ctr1- <dig> could be due to greater ethylene evolution than the wild type , as supported by ethylene evolution .
these results suggest that the ecr2- <dig> allele conferred a minor growth inhibition throughout development.
as compared with the wild type , etiolated ecr2- <dig> seedlings showed no alteration in ethylene sensitivity.
ctr1-independent but not -dependent etr <dig> receptor signaling occurs in ecr2- <dig> ctr1-10
silver treatment prevents the inhibition of etr <dig> receptor signaling by ethylene  <cit> .
silver treatment reversed the ethylene effect on growth of ctr1- <dig> but not ecr2- <dig> ctr1- <dig> seedlings , so etr <dig> receptor signaling was prevented in ecr2- <dig> ctr1- <dig> but partly mediated in ctr1- <dig>  we evaluated whether etr <dig> receptor signaling could occur in ecr2- <dig> ctr1- <dig>
ethylene-insensitive etr1- <dig> and etr1- <dig> mediate receptor signaling by different mechanisms: the former does not require reversion-to-ethylene sensitivity <dig> , whereas the latter does  <cit> .
because of their dominant nature, we used etr1- <dig> and etr1- <dig> mutants to evaluate etr <dig> receptor signaling.
in the absence of ethylene treatment, etiolated etr1- <dig> and etr1- <dig> seedlings produced a long hypocotyl, and the ctr1- <dig> mutation moderately reduced the seedling hypocotyl elongation in each allele; as expected, etr1- <dig> ecr2- <dig> ctr1- <dig> and etr1- <dig> ecr2- <dig> ctr1- <dig> seedlings were short, with a hypocotyl length similar to ecr2- <dig> ctr1- <dig> seedlings .
consistently, at the adult stage, the rosette was larger for etr1- <dig> ctr1- <dig> and etr1- <dig> ctr1- <dig> than etr1- <dig> ecr2- <dig> ctr1- <dig> and etr1- <dig> ecr2- <dig> ctr1- <dig> plants, respectively .
we scored the ethylene response in these genotypes by measuring erf <dig> expression: the erf <dig> level was greater in etr1- <dig> ctr1- <dig> and etr1- <dig> ctr1- <dig> than the wild type and lower than in etr1- <dig> ecr2- <dig> ctr1- <dig> and etr1- <dig> ecr2- <dig> ctr1- <dig> .
etr <dig> receptor signaling may be prevented by ecr2- <dig> ctr1- <dig> but little affected by ctr1- <dig>
etr <dig> receptor signaling can be mediated via the n-terminal portion independent of ctr <dig>  <cit> , so we evaluated whether ecr <dig> could act in the ctr1-independent pathway.
with ethylene treatment, ctr1- <dig> seedlings expressing the etr1p:etr1-1
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transgene that encodes the etr1-1 n terminus  were ethylene insensitive and produced a long hypocotyl  <cit>  .
interestingly, the phenotype of ethylene growth inhibition in ecr2- <dig> ctr1- <dig> was rescued by the transgene, and etr1p:etr1-1
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ecr2- <dig> ctr1- <dig> seedlings did not show the phenotype of ethylene-inhibited growth .
consistently, etr1p:etr1-1
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expression greatly rescued rosette growth in ecr2- <dig> ctr1- <dig> and ctr1- <dig> .
the expression of the transgene attenuated erf <dig> levels in ecr2- <dig> ctr1- <dig> and ctr1- <dig> .
expression of etr1p:etr1-1
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in the transformation lines was confirmed by qrt-pcr with etr1-1–specific primers, and etr1- <dig> expression was detectable only in genotypes that carried the allele or transgene .
these data suggest that the ecr2- <dig> allele prevented the receptor signaling with the full-length etr1- <dig> and etr1- <dig> but not etr1-1 n terminus in ctr1- <dig>  therefore, ecr <dig> may be involved in ctr1-dependent but not -independent receptor signaling in ctr1- <dig>  given that the signaling of full-length etr <dig> but not etr <dig> n terminus is prevented by kinase-defective ctr <dig> isoforms  <cit> , ecr <dig> could be involved in part in ctr <dig> kinase activity or protein stability.
ctr1- <dig> is not associated with the ethylene receptors, and the mutant shows a weak constitutive ethylene response.
we previously hypothesized that mediation of ethylene receptor signaling in ctr1- <dig> was independent of ctr <dig>  <cit> .
the possibility that ctr1- <dig> could mediate in part the receptor signaling, for a weak mutant phenotype, should be considered.
given that ecr <dig> is involved in ctr1-dependent ethylene receptor signaling, we expected that ecr2- <dig> would have little effect on ctr1- <dig> if ctr1- <dig> did not mediate the ethylene receptor signaling.
we crossed ecr2- <dig> ctr1- <dig> with ctr1- <dig>  and the resulting f <dig> plants were genotyped for the ctr <dig> allele.
all f <dig> plants that showed the typical constitutive ethylene response phenotype were ctr1- <dig> or ctr1- <dig> ctr1- <dig>  so they all carried the homozygous ecr2- <dig> allele and ecr2- <dig> did not enhance the ctr1- <dig> mutant phenotype.
the mutant ecr2- <dig> ctr1- <dig> ctr1- <dig> was selfed, and the resulting progenies with the homozygous ctr1- <dig> allele were ecr2- <dig> ctr1- <dig>
the length of etiolated ecr2- <dig> ctr1- <dig> seedlings was only  <dig> ± <dig>  mm  shorter than that of ctr1- <dig> seedlings , which is in line with the result showing that the ecr2- <dig> allele conferred minor seedling growth inhibition  but not increased ethylene sensitivity .
light-grown seedlings of ctr1- <dig> and ecr2- <dig> ctr1- <dig> were phenotypically similar, with a shorter root and smaller cotyledons than the wild type  .
consistently, the rosette was larger for the wild type than ctr1- <dig> and ecr <dig> ctr1- <dig> plants .
therefore, the ecr2- <dig> allele may have little effect on the ethylene response phenotype in ctr1- <dig>  we quantified the ethylene response for the two mutants.
erf <dig> expression was identical in ctr1- <dig> and ecr2- <dig> ctr1- <dig>  and higher than in the wild type .
both ctr1- <dig> and ecr2- <dig> ctr1- <dig> had the same senescence phenotype, and their chlorophyll a content was identical before  and after  the test .
the ecr2- <dig> allele had little effect on ctr1- <dig> ethylene response, which suggests that ethylene receptor signaling in ctr1- <dig> was predominantly mediated by a pathway independent of ctr <dig>
genetic analysis of effect of ein3- <dig> and eil1- <dig> on the ethylene response in ecr2- <dig> ctr1-10
the present data suggested the involvement of ecr <dig> in ethylene receptor signaling mediated by ctr <dig>  ethylene insensitive <dig>  acts downstream of ctr <dig>  and ein <dig> loss-of-function mutation may rescue the ecr2- <dig> ctr1- <dig> mutant phenotype.
genetic analyses for the effects of ein <dig> on ecr2- <dig> ctr1- <dig> were not successful because ein <dig> is tightly linked with ctr <dig> and ecr <dig> remains to be cloned.
ein <dig> and ein3-like <dig>  are the transcription factors that direct the expression of ethylene-responsive genes, and ein <dig> loss-of-function mutations confer ethylene insensitivity  <cit> .
we used genetic analyses to evaluate whether ecr <dig> acts upstream of ein3/eil <dig>  to obtain ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> mutants, ein3- <dig> and eil1- <dig> were each genetically crossed with ecr2- <dig> ctr1- <dig>  and the resulting f <dig> individuals carrying ein3- <dig> ctr1- <dig> and eil1- <dig> ctr1- <dig> were identified by genotyping.
all f <dig> generations with the ein3- <dig> ctr1- <dig> and eil1- <dig> ctr1- <dig> genotypes showed relatively normal growth.
the f <dig> individuals were each genetically crossed with ecr2- <dig> ctr1- <dig> to test the presence of the ecr2- <dig> allele, and the individuals that were ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> would give a 1: <dig> segregation  in the resulting f <dig> generation.
verified by chi-square test for  <dig> independent test crossings, we isolated ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> .
without ethylene treatment, ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> seedlings produced a long seedling hypocotyl  as compared with ecr2- <dig> ctr1- <dig> seedlings .
of note, ein3- <dig> but not eil1- <dig> seedlings were ethylene-insensitive, and ethylene treatment inhibited eil1- <dig> but not ein3- <dig> seedling growth.
consistently, the ein3- <dig> allele prevented ethylene-inhibited growth of seedling hypocotyls in ecr2- <dig> ctr1- <dig> whereas eil1- <dig> did not .
at the adult stage, ein3- <dig> and eil1- <dig> alleles each rescued ecr2- <dig> ctr1- <dig> rosette growth to a great extent, and rosettes were larger for ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> than ecr2- <dig> ctr1- <dig> plants .
the ethylene response in each triple mutant was quantified by leaf senescence and erf <dig> expression.
the leaf senescence phenotype was weaker in ein3- <dig>  eil1- <dig> and ein3- <dig> ecr2- <dig> ctr1- <dig> than ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> plants , which was consistent with chlorophyll a content .
without ethylene treatment, the erf <dig> level in ecr2- <dig> ctr1- <dig> was highly reduced with the respective addition of ein3- <dig> and eil1- <dig> alleles, and ein3- <dig>  eil1- <dig>  ein3- <dig> ecr2- <dig> ctr1- <dig> and eil1- <dig> ecr2- <dig> ctr1- <dig> showed identical erf <dig> expression .
the present data support that ecr <dig> acts in ethylene signal transduction upstream of the transcription factors ein <dig> and eil <dig>  the eil1- <dig> allele did not prevent the ethylene-inhibited seedling growth or leaf senescence in ecr2- <dig> ctr1- <dig>  which indicates that functions of ein <dig> and eil <dig> differ, with a larger role for ein <dig> than eil <dig> in ethylene responses.
ebf <dig> and ebf <dig> overexpression rescues ecr2- <dig> ctr1- <dig> phenotype
ein3-binding f-box <dig>  and ebf <dig> are f-box proteins involved in the ubiquitination of ein <dig> and eil <dig> to mediate their degradation by the 26s proteosome  <cit> .
excess ebf <dig> and ebf <dig> reduces ein <dig> and eil <dig> levels and suppresses the ethylene response  <cit> .
the argument that ecr <dig> acts in the ethylene signal transduction pathway upstream of the transcription factors ein <dig> and eil <dig> can be tested by examining whether overexpression of ebf <dig> and ebf <dig> rescues the ecr2- <dig> ctr1- <dig> phenotype.
ebf <dig> and ebf <dig> were each expressed under the regulation of the constitutive cauliflower mosaic virus 35s promoter in ecr2- <dig> ctr1- <dig> and ctr1- <dig>  as expected, with ethylene treatment, hypocotyls were longer for etiolated seedlings of 35s:ebf <dig> ecr2- <dig> ctr1- <dig> and 35s:ebf <dig> ecr2- <dig> ctr1- <dig>  than the wild type , as well as longer for the strong allele ctr1- <dig> expressing the transgene than ctr1- <dig> .
consistently, ethylene treatment inhibited the cotyledon expansion in the wild type  but not the transformation lines in light-grown seedlings .
at the adult stage, the expression of each transgene largely rescued the growth inhibition phenotype in ecr2- <dig> ctr1- <dig> and ctr1- <dig>  rosettes were slightly smaller for 35s:ebf <dig> ecr2- <dig> ctr1- <dig> lines than the wild type, but those for 35s:ebf <dig> ecr2- <dig> ctr1- <dig> lines and the wild type were similar in size .
confirmation of the expression of the transgenes by qrt-pcr revealed lower ebf <dig> level in 35s:ebf <dig> ctr1- <dig> than in 35s:ebf <dig> ecr <dig> ctr1- <dig> lines .
the lower ebf <dig> expression could be associated with the stronger phenotype of rosette growth inhibition in 35s:ebf <dig> ctr1- <dig> than 35s:ebf <dig> ecr2- <dig> ctr1- <dig>  in contrast, ebf <dig> level was higher in 35s:ebf <dig> ctr1- <dig> than 35s:ebf <dig> ecr2- <dig> ctr1- <dig> lines .
we also examined other aspects of the ethylene response affected by the transgenes.
erf <dig> expression was greater in ecr2- <dig> ctr1- <dig> and ctr1- <dig> than the wild type; the expression of each transgene largely reduced erf <dig> levels .
of note, erf <dig> expression was greater in 35s:ebf <dig> ctr1- <dig> than 35s:ebf <dig> ecr2- <dig> ctr1- <dig> lines, likely because of its lower ebf <dig> expression .
consistent with the suppression of ecr2- <dig> ctr1- <dig> by ein3- <dig> and eil1- <dig>  the respective elevation in ebf <dig> and ebf <dig> levels in ecr2- <dig> ctr1- <dig> rescued the inhibition in mutant growth and reduced erf <dig> levels, which supports that ecr <dig> acts upstream of the transcriptions factors ein <dig> and eil <dig> in the ethylene signal transduction pathway.
discussion
arabidopsis ethylene signaling is negatively regulated by ethylene receptors and the raf-like protein ctr <dig>  <cit> .
the biochemical nature of receptor signaling is unknown, as are the underlying mechanisms by which ctr <dig> is activated  <cit> .
a genetic screen for enhancers facilitates the isolation of genes or mutations that play a role in the same biological process as the known mutation.
given that the ctr1- <dig> mutation does not disrupt the ctr <dig> orf and that ctr1- <dig> is a weak allele, an enhancer screen for ctr1- <dig> could lead to isolation of components involved in ctr1-mediated ethylene receptor signaling.
in this study, we reported the isolation of ecr2- <dig> as an enhancer mutation for ctr1- <dig>  with a relative weak constitutive ethylene response phenotype for ctr1- <dig> and a strong phenotype for ecr2- <dig> ctr1- <dig>  the mutations ecr2- <dig> and ctr1- <dig> synergistically facilitated ethylene signaling.
the assumption that ctr <dig> level in ctr1- <dig> could be reduced needs to be verified biochemically.
nevertheless, extensive studies suggest that for a common transcript, a long 5′-utr can be translationally inhibitory as compared with a short one  <cit> .
conceivably, the t-dna insertion that increases the 5′-utr length may add structures of higher order and uorfs to inhibit the translation of ctr <dig>  thus reducing ctr <dig> level, so that ctr1- <dig> shows a weak constitutive ethylene response phenotype.
we considered  <dig> scenarios explaining the synergistic effect of ecr2- <dig> and ctr1-10: 1) ecr <dig> could be required in part for ctr <dig> activity or the protein stability, 2) ecr <dig> and ctr <dig> act in different pathways, or 3) ecr <dig> could be a signaling molecule acting with ctr <dig> to suppress the ethylene signaling.
in the first scenario, ctr <dig> level, and possibly activity, in ctr1- <dig> is presumably reduced to a level requiring ecr <dig> to suppress the ethylene signaling.
the nature of ctr1- <dig> and ctr1- <dig> mutations is distinct: ctr <dig> level could be reduced in ctr1- <dig> and the ctr1- <dig> protein is not associated with the ethylene receptors  <cit> .
the docking of kinase-defective ctr <dig> isoforms at the hk domain of etr <dig> prevents receptor signaling to an alternative ctr1-independent pathway.
in ctr1- <dig>  the ethylene receptors are free from association with ctr1-8; thus, the receptor signaling is mediated to the alternative pathway and the mutant shows a weak phenotype  <cit> .
conceivably, in ecr2- <dig> ctr1- <dig>  the activity of ctr <dig> docking at the ethylene receptors could be greatly reduced, and ethylene receptor signaling that is mediated by ctr <dig> as well as the alternative pathway was prevented.
in contrast, the ctr1- <dig> protein does not dock at the ethylene receptors and the receptor signaling can be mediated by the alternative, ctr1-independent pathway; thus, ecr2- <dig> had little effect on ctr1- <dig> phenotype.
in line with these results, our data showing that ecr2- <dig> prevented etr1- <dig> and etr1- <dig> but not etr1-11- <dig> receptor signaling in ctr1- <dig> also support that ecr <dig> is involved in the ethylene receptor signaling that is dependent on ctr <dig>
an alternative explanation for the distinct effects of ecr2- <dig> on ctr1- <dig> and ctr1- <dig> could arise from a role of ecr <dig> in ctr <dig> stability.
in this scenario, very small amount of the ctr1- <dig> protein that could associate with the ethylene receptors could have a higher turnover rate in the absence of ecr2; meanwhile, ctr1- <dig> reserved in the soluble fraction could be continuously recruited to the receptors and mediate the receptor signaling.
contrarily, in ctr1- <dig>  ctr <dig> level is highly reduced without ecr <dig> and the receptor signaling is prevented.
however, this scenario is not consistent with the argument that the ethylene receptor signaling can be mediated to an alternative pathway that is independent of ctr <dig>  in other words, if ecr <dig> were involved in ctr <dig> stability, the ethylene receptor signaling that was mediated independent of ctr <dig> still occurred and the ecr2- <dig> allele would have little effect on the degree of ctr1- <dig> ethylene responses.
CONCLUSIONS
ctr <dig> is presumably activated by the ethylene receptors, and the mechanism is unknown  <cit> .
little is known about the presence of any other components that are involved in the ethylene receptor signaling to ctr <dig>  the present data suggest that ecr <dig> is a component involved in part in ctr1-mediated ethylene receptor signaling, which indicates a regulatory mechanism for the receptor signaling.
we favor the argument that ctr <dig> activity but not level was affected by ecr2- <dig> in ctr1- <dig>  with limited sequence information for the 598-kb region that contains ecr <dig>  the cloning of ecr <dig> is currently hampered.
complementation of ecr2- <dig> by a large dna fragment that contains ecr <dig> is ongoing.
however, with repeated sequences, transposons, retrotransposons, and sequence gaps in this region, cloning of a large dna fragment is still highly challenging.
nevertheless, the cloning of ecr <dig> will advance our knowledge of ethylene signaling that involves ctr <dig> activation.

 ethylene is a gaseous hormone regulating many aspects of plant growth and development. the dicotyledonous model plant arabidopsis has five ethylene receptors that physically act at the endoplasmic reticulum with the raf-like protein constitutive triple-response to negatively regulate ethylene signaling. in the absence of ethylene, the receptor signal output is mediated by an unknown mechanism to activate ctr and ctr is presumably activated to phosphorylate the downstream signaling component ethylene insensitive. phosphorylated ein stays at the er and cannot induce the ethylene response. with ethylene binding to ethylene receptors, the receptor signal output is prevented and ctr is not activated. unphosphorylated ein undergoes proteolytic cleavage by an unknown mechanism to produce a c-terminal fragment, which enters the nucleus to induce the ethylene response. targets of the ein c terminus remain to be identified. the transcription factors ein and ein3-like direct the expression of ethylene-responsive genes, and ein c-terminus–induced ethylene responses are prevented in the ein3- loss-of-function mutant. conceivably, the ein c terminus could mediate the ethylene signaling to ein and eil the ethylene receptors are structurally similar to histidine kinases of prokaryotic two-component modules, and studies have revealed the domain functions of the ethylene receptors. the n terminus has three or four transmembrane domains that bind a copper cofactor for ethylene binding and are required for localization at the er. following the tms is the gaf domain for non-covalent receptor interaction to mediate inter-receptor signaling. the c-terminal portion is the hk domain, which is believed to function in the receptor signal output via direct interaction with the ctr n terminus. ctr has serine/threonine kinase activity, and the ethylene response is inversely associated with ctr kinase activity. these studies suggest that the hk domain mediates ethylene receptor signaling to the ctr n terminus, thus activating ctr to suppress the ethylene response, although the underlying biochemical mechanisms are elusive. recent studies suggest that the hk domain can be dispensable to ethylene receptor signal output and that ctr is not the only component mediating the signaling. mutations that delete ethylene response hk and receiver domains have little effect on the receptor signal output, and expression of the truncated etr11- isoform that lacks the site for interacting with ctr rescues the ctr loss-of-function mutant phenotype. conceivably, the etr receptor signal output can be mediated via the n terminus to an alternative pathway independent of ctr. of note, etr receptor signaling in kinase-defective ctr mutants occurs only when hk-domain–lacking etr isoforms are expressed. a kinase-defective ctr isoform may dock at the etr hk domain and actively prevent etr n-terminal signaling to the ctr1-independent alternative pathway. the ctr1- mutation results from a t-dna insertion at the 5′-untranslated region of ctr. the 5′-utr of a mrna may be highly structured and contain upstream augs and internal ribosome entry sites that can affect the translation. with increased upstream open reading frames and a highly structured nature, a long 5′-utr can be translationally inhibitory. with a higher ctr level in ctr1- than in the wild type, the t-dna insertion may substantially increase the 5′-utr length of the ctr transcript to affect translation efficiency, thus reducing the ctr level and thus activity as compared with the wild type. the underlying biochemical mechanisms of ctr activation by the ethylene receptors remain to be unraveled. mutations that enhance the constitutive ethylene response in ctr1- could have a role involving ctr activation, protein stability or ethylene signaling. here we report that the loss-of-function mutation of enhancing ctr1- ethylene response enhanced the ethylene response in ctr1- comparable to the strong allele ctr1- and the ethylene-treated wild type. results from extensive genetic and transformation studies suggested that ecr acts downstream of the ethylene receptors and upstream of the positive ethylene response regulators ein and eil we discuss possible roles for ecr in the negative regulation of the ethylene response.  ctr1- is a weak allele the ctr1- mutation results from a t-dna insertion at the 5′-utr, and the mutant shows a weak constitutive ethylene response phenotype. the nature of the mutation that causes the weak phenotype remains to be determined. given that the t-dna inserts at the 5′-utr, we expected that the t-dna fragment is transcribed as part of the 5′-utr of ctr1- mrna. indeed, rt-pcr with primers pairing the 5′-utr and t-dna sequences showed the presence of a chimeric cdna fragment containing the sequence of the ctr1- 5′-utr and the t-dna. for the same gene, a longer 5′-utr can be translationally more inhibitory than a shorter one. with a -kb t-dna at the 5′-utr, we hypothesized that ctr1- could produce a lower level of ctr than the wild type, for a weak constitutive ethylene response phenotype. ethylene inhibits hypocotyl growth and the ethylene response can be quantified by measurement of hypocotyls. hypocotyls were shorter for ctr1- than wild-type seedlings over a wide range of ethylene concentrations. when growth for wild-type and ctr1- seedlings are normalized to growth with no added ethylene, it becomes apparent that ctr1- seedlings are more sensitive to ethylene with a shift from μl l- for wild-type to μl l- for the mutant. these data suggest increased ethylene sensitivity with the ctr1- mutation. genetic screen for ctr1- enhancer mutations to isolate components that could be involved in ctr1-mediated ethylene receptor signaling, we performed an enhancer screen for ctr1- alleles that enhanced the ctr1- mutant phenotype were designated enhancing ctr1- ethylene response. here we describe the isolation of ecr2- ctr1- without ethylene treatment, etiolated seedlings of ecr2- ctr1- and the strong allele ctr1- were identical in phenotype and hypocotyl length; ctr1- seedlings were longer than the two genotypes and shorter than the wild type. as expected, ethylene treatment inhibited the seedling growth in the wild type, ctr1- and ecr2- ctr1-. of note, ecr2- ctr1- seedlings already showed a constitutive ethylene response phenotype, with a short hypocotyl, so that the effect of ethylene on the seedling growth inhibition was minor. the silver ion ag may bind etr and prevent ethylene inhibition of etr receptor signaling. silver treatment largely prevented ethylene-induced inhibition of seedling hypocotyl growth in wild-type and ctr1- but not ecr2- ctr1- seedlings. of note, the seedling hypocotyls were longer for silver-treated than non-silver–treated ecr2- ctr1- seedlings, regardless of ethylene treatment; the difference was minor and could be of little biological significance. these results suggest that etr receptor signaling that was mediated in part in ctr1- was prevented in ecr2- ctr1- aminoethoxyvinylglycine prevents the biosynthesis of the immediate ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic, and the treatment can reduce endogenous ethylene production. avg treatment did not rescue the reduced growth of ecr2- ctr1- seedlings, so ecr2- did not enhance the production of ethylene to cause reduced growth. ethylene overproducer is a negative regulator of the acc-synthesing enzyme acc synthase and the eto1- allele produces a higher level of acc, and therefore ethylene, than does the wild type. as a control, eto1- seedlings showed a short hypocotyl, and growth was rescued with avg treatment; avg treatment in this study was sufficient to prevent endogenous ethylene production. we examined the growth inhibition phenotype at other developmental stages. grown under light, ethylene treatment inhibited cotyledon expansion and root elongation in wild-type seedlings. seedlings of ctr1- ctr1- and ecr2- ctr1- showed the growth inhibition phenotype regardless of ethylene treatment. at the adult stage, rosettes were larger for the wild type than the three mutants, with ctr1- and ecr2- ctr1- being phenotypically similar and both producing a smaller rosette than ctr1-. we examined alterations in other aspects of the ethylene response. leaves of the wild type, ctr1- ctr1- and ecr2- ctr1- showed the senescence phenotype to various degrees 5 days after detachment. detached leaves of the mutants showed a similar chlorophyll a level, with a similar or slightly higher level in ctr1- than ctr1- and ecr2- ctr1-. ethylene response factor expression is directed by ein and the expression is associated with degrees of ethylene response. erf expression was slightly higher in ctr1- than the wild type and highly induced in ctr1- and ecr2- ctr1- without ethylene treatment. ctr levels in ctr1- ctr1- and ecr2- ctr1- were identical and higher than in the wild type. the ecr2- allele did not affect ctr expression in ctr1- ecr2- is a recessive, loss-of-function mutation to genetically evaluate whether the effect of the ecr2- mutation on the ctr1- mutant phenotype is associated with single or multiple alleles, we crossed ecr2- ctr1- with the wild type and ctr1- in the f generation of the wild type cross, individuals were scored, and only showed the ecr2- ctr1- growth inhibition phenotype. in the f generation of the ctr1- cross, individuals were scored, and showed the ecr2- ctr1- growth inhibition phenotype. results from both genetic analyses suggested that the ecr2- loss-of-function mutation was recessive and intergenic to ctr1- the independent segregation of ecr2- and ctr1- suggested that ecr and ctr are in distinct linkage groups. we attempted to clone ecr by map-based cloning and mapped ecr to a 598-kb region spanning the centromere at chromosome, which agreed with ecr and ctr being in distinct linkage groups. however, we did not obtain more recombinants to advance the mapping to a narrower region. this region contains sequence gaps and fragments with repeated sequences, retrotransposon, transposase, and transposable elements. we sequenced annotated genes but did not identify any mutation. among the genes, t-dna insertion mutants for at2g07981and at2g were each genetically crossed with ctr1- to test whether any of these was ecr and these mutations did not enhance the ctr1- phenotype. the mutation that affects ecr remained to be identified and we attempted to isolate the ecr2- single mutant. the f seedlings from the genetic crossing of ecr2- ctr1- and the wild type were crossed with ctr1- and the individual that produced the ecr2- ctr1- mutant phenotype in the f generation with a 1: segregation ratio was considered the ecr2- mutant. we isolated ecr2- and results from independent crossings with ctr1- showed a 1: segregation. grown in dark, ecr2- seedlings produced a shorter hypocotyl than did the wild type at low ethylene concentrations; without ethylene treatment, the height of ecr2- seedlings was ±  mm shorter than that of wild-type seedlings. when this data was normalized, we found that wild-type and ecr2- seedlings had indistinguishable sensitivity to ethylene. although the relative hypocotyl length was longer for ecr2- seedlings than wild-type seedlings with μl l- ethylene, the difference was small and could be of little biological significance. at the adult stage, the rosette was smaller for ecr2- than the wild type. we excluded the possibility that the mutant phenotype of ecr2- and ecr2- ctr1- could be due to greater ethylene evolution than the wild type, as supported by ethylene evolution. these results suggest that the ecr2- allele conferred a minor growth inhibition throughout development. as compared with the wild type, etiolated ecr2- seedlings showed no alteration in ethylene sensitivity. ctr1-independent but not -dependent etr receptor signaling occurs in ecr2- ctr1-10 silver treatment prevents the inhibition of etr receptor signaling by ethylene. silver treatment reversed the ethylene effect on growth of ctr1- but not ecr2- ctr1- seedlings, so etr receptor signaling was prevented in ecr2- ctr1- but partly mediated in ctr1- we evaluated whether etr receptor signaling could occur in ecr2- ctr1- ethylene-insensitive etr1- and etr1- mediate receptor signaling by different mechanisms: the former does not require reversion-to-ethylene sensitivity, whereas the latter does. because of their dominant nature, we used etr1- and etr1- mutants to evaluate etr receptor signaling. in the absence of ethylene treatment, etiolated etr1- and etr1- seedlings produced a long hypocotyl, and the ctr1- mutation moderately reduced the seedling hypocotyl elongation in each allele; as expected, etr1- ecr2- ctr1- and etr1- ecr2- ctr1- seedlings were short, with a hypocotyl length similar to ecr2- ctr1- seedlings. consistently, at the adult stage, the rosette was larger for etr1- ctr1- and etr1- ctr1- than etr1- ecr2- ctr1- and etr1- ecr2- ctr1- plants, respectively. we scored the ethylene response in these genotypes by measuring erf expression: the erf level was greater in etr1- ctr1- and etr1- ctr1- than the wild type and lower than in etr1- ecr2- ctr1- and etr1- ecr2- ctr1-. etr receptor signaling may be prevented by ecr2- ctr1- but little affected by ctr1- etr receptor signaling can be mediated via the n-terminal portion independent of ctr, so we evaluated whether ecr could act in the ctr1-independent pathway. with ethylene treatment, ctr1- seedlings expressing the etr1p:etr1-1 1-349 transgene that encodes the etr1-1 n terminus were ethylene insensitive and produced a long hypocotyl. interestingly, the phenotype of ethylene growth inhibition in ecr2- ctr1- was rescued by the transgene, and etr1p:etr1-1 1-349 ecr2- ctr1- seedlings did not show the phenotype of ethylene-inhibited growth. consistently, etr1p:etr1-1 1-349 expression greatly rescued rosette growth in ecr2- ctr1- and ctr1-. the expression of the transgene attenuated erf levels in ecr2- ctr1- and ctr1-. expression of etr1p:etr1-1 1-349 in the transformation lines was confirmed by qrt-pcr with etr1-1–specific primers, and etr1- expression was detectable only in genotypes that carried the allele or transgene. these data suggest that the ecr2- allele prevented the receptor signaling with the full-length etr1- and etr1- but not etr1-1 n terminus in ctr1- therefore, ecr may be involved in ctr1-dependent but not -independent receptor signaling in ctr1- given that the signaling of full-length etr but not etr n terminus is prevented by kinase-defective ctr isoforms, ecr could be involved in part in ctr kinase activity or protein stability. ctr1- is not associated with the ethylene receptors, and the mutant shows a weak constitutive ethylene response. we previously hypothesized that mediation of ethylene receptor signaling in ctr1- was independent of ctr. the possibility that ctr1- could mediate in part the receptor signaling, for a weak mutant phenotype, should be considered. given that ecr is involved in ctr1-dependent ethylene receptor signaling, we expected that ecr2- would have little effect on ctr1- if ctr1- did not mediate the ethylene receptor signaling. we crossed ecr2- ctr1- with ctr1- and the resulting f plants were genotyped for the ctr allele. all f plants that showed the typical constitutive ethylene response phenotype were ctr1- or ctr1- ctr1- so they all carried the homozygous ecr2- allele and ecr2- did not enhance the ctr1- mutant phenotype. the mutant ecr2- ctr1- ctr1- was selfed, and the resulting progenies with the homozygous ctr1- allele were ecr2- ctr1- the length of etiolated ecr2- ctr1- seedlings was only ±  mm shorter than that of ctr1- seedlings, which is in line with the result showing that the ecr2- allele conferred minor seedling growth inhibition but not increased ethylene sensitivity. light-grown seedlings of ctr1- and ecr2- ctr1- were phenotypically similar, with a shorter root and smaller cotyledons than the wild type. consistently, the rosette was larger for the wild type than ctr1- and ecr ctr1- plants. therefore, the ecr2- allele may have little effect on the ethylene response phenotype in ctr1- we quantified the ethylene response for the two mutants. erf expression was identical in ctr1- and ecr2- ctr1- and higher than in the wild type. both ctr1- and ecr2- ctr1- had the same senescence phenotype, and their chlorophyll a content was identical before and after the test. the ecr2- allele had little effect on ctr1- ethylene response, which suggests that ethylene receptor signaling in ctr1- was predominantly mediated by a pathway independent of ctr genetic analysis of effect of ein3- and eil1- on the ethylene response in ecr2- ctr1-10 the present data suggested the involvement of ecr in ethylene receptor signaling mediated by ctr ethylene insensitive acts downstream of ctr and ein loss-of-function mutation may rescue the ecr2- ctr1- mutant phenotype. genetic analyses for the effects of ein on ecr2- ctr1- were not successful because ein is tightly linked with ctr and ecr remains to be cloned. ein and ein3-like are the transcription factors that direct the expression of ethylene-responsive genes, and ein loss-of-function mutations confer ethylene insensitivity. we used genetic analyses to evaluate whether ecr acts upstream of ein3/eil to obtain ein3- ecr2- ctr1- and eil1- ecr2- ctr1- mutants, ein3- and eil1- were each genetically crossed with ecr2- ctr1- and the resulting f individuals carrying ein3- ctr1- and eil1- ctr1- were identified by genotyping. all f generations with the ein3- ctr1- and eil1- ctr1- genotypes showed relatively normal growth. the f individuals were each genetically crossed with ecr2- ctr1- to test the presence of the ecr2- allele, and the individuals that were ein3- ecr2- ctr1- and eil1- ecr2- ctr1- would give a 1: segregation in the resulting f generation. verified by chi-square test for independent test crossings, we isolated ein3- ecr2- ctr1- and eil1- ecr2- ctr1-. without ethylene treatment, ein3- ecr2- ctr1- and eil1- ecr2- ctr1- seedlings produced a long seedling hypocotyl as compared with ecr2- ctr1- seedlings. of note, ein3- but not eil1- seedlings were ethylene-insensitive, and ethylene treatment inhibited eil1- but not ein3- seedling growth. consistently, the ein3- allele prevented ethylene-inhibited growth of seedling hypocotyls in ecr2- ctr1- whereas eil1- did not. at the adult stage, ein3- and eil1- alleles each rescued ecr2- ctr1- rosette growth to a great extent, and rosettes were larger for ein3- ecr2- ctr1- and eil1- ecr2- ctr1- than ecr2- ctr1- plants. the ethylene response in each triple mutant was quantified by leaf senescence and erf expression. the leaf senescence phenotype was weaker in ein3- eil1- and ein3- ecr2- ctr1- than ecr2- ctr1- and eil1- ecr2- ctr1- plants, which was consistent with chlorophyll a content. without ethylene treatment, the erf level in ecr2- ctr1- was highly reduced with the respective addition of ein3- and eil1- alleles, and ein3- eil1- ein3- ecr2- ctr1- and eil1- ecr2- ctr1- showed identical erf expression. the present data support that ecr acts in ethylene signal transduction upstream of the transcription factors ein and eil the eil1- allele did not prevent the ethylene-inhibited seedling growth or leaf senescence in ecr2- ctr1- which indicates that functions of ein and eil differ, with a larger role for ein than eil in ethylene responses. ebf and ebf overexpression rescues ecr2- ctr1- phenotype ein3-binding f-box and ebf are f-box proteins involved in the ubiquitination of ein and eil to mediate their degradation by the 26s proteosome. excess ebf and ebf reduces ein and eil levels and suppresses the ethylene response. the argument that ecr acts in the ethylene signal transduction pathway upstream of the transcription factors ein and eil can be tested by examining whether overexpression of ebf and ebf rescues the ecr2- ctr1- phenotype. ebf and ebf were each expressed under the regulation of the constitutive cauliflower mosaic virus 35s promoter in ecr2- ctr1- and ctr1- as expected, with ethylene treatment, hypocotyls were longer for etiolated seedlings of 35s:ebf ecr2- ctr1- and 35s:ebf ecr2- ctr1- than the wild type, as well as longer for the strong allele ctr1- expressing the transgene than ctr1-. consistently, ethylene treatment inhibited the cotyledon expansion in the wild type but not the transformation lines in light-grown seedlings. at the adult stage, the expression of each transgene largely rescued the growth inhibition phenotype in ecr2- ctr1- and ctr1- rosettes were slightly smaller for 35s:ebf ecr2- ctr1- lines than the wild type, but those for 35s:ebf ecr2- ctr1- lines and the wild type were similar in size. confirmation of the expression of the transgenes by qrt-pcr revealed lower ebf level in 35s:ebf ctr1- than in 35s:ebf ecr ctr1- lines. the lower ebf expression could be associated with the stronger phenotype of rosette growth inhibition in 35s:ebf ctr1- than 35s:ebf ecr2- ctr1- in contrast, ebf level was higher in 35s:ebf ctr1- than 35s:ebf ecr2- ctr1- lines. we also examined other aspects of the ethylene response affected by the transgenes. erf expression was greater in ecr2- ctr1- and ctr1- than the wild type; the expression of each transgene largely reduced erf levels. of note, erf expression was greater in 35s:ebf ctr1- than 35s:ebf ecr2- ctr1- lines, likely because of its lower ebf expression. consistent with the suppression of ecr2- ctr1- by ein3- and eil1- the respective elevation in ebf and ebf levels in ecr2- ctr1- rescued the inhibition in mutant growth and reduced erf levels, which supports that ecr acts upstream of the transcriptions factors ein and eil in the ethylene signal transduction pathway. discussion arabidopsis ethylene signaling is negatively regulated by ethylene receptors and the raf-like protein ctr. the biochemical nature of receptor signaling is unknown, as are the underlying mechanisms by which ctr is activated. a genetic screen for enhancers facilitates the isolation of genes or mutations that play a role in the same biological process as the known mutation. given that the ctr1- mutation does not disrupt the ctr orf and that ctr1- is a weak allele, an enhancer screen for ctr1- could lead to isolation of components involved in ctr1-mediated ethylene receptor signaling. in this study, we reported the isolation of ecr2- as an enhancer mutation for ctr1- with a relative weak constitutive ethylene response phenotype for ctr1- and a strong phenotype for ecr2- ctr1- the mutations ecr2- and ctr1- synergistically facilitated ethylene signaling. the assumption that ctr level in ctr1- could be reduced needs to be verified biochemically. nevertheless, extensive studies suggest that for a common transcript, a long 5′-utr can be translationally inhibitory as compared with a short one. conceivably, the t-dna insertion that increases the 5′-utr length may add structures of higher order and uorfs to inhibit the translation of ctr thus reducing ctr level, so that ctr1- shows a weak constitutive ethylene response phenotype. we considered scenarios explaining the synergistic effect of ecr2- and ctr1-10: 1) ecr could be required in part for ctr activity or the protein stability, 2) ecr and ctr act in different pathways, or 3) ecr could be a signaling molecule acting with ctr to suppress the ethylene signaling. in the first scenario, ctr level, and possibly activity, in ctr1- is presumably reduced to a level requiring ecr to suppress the ethylene signaling. the nature of ctr1- and ctr1- mutations is distinct: ctr level could be reduced in ctr1- and the ctr1- protein is not associated with the ethylene receptors. the docking of kinase-defective ctr isoforms at the hk domain of etr prevents receptor signaling to an alternative ctr1-independent pathway. in ctr1- the ethylene receptors are free from association with ctr1-8; thus, the receptor signaling is mediated to the alternative pathway and the mutant shows a weak phenotype. conceivably, in ecr2- ctr1- the activity of ctr docking at the ethylene receptors could be greatly reduced, and ethylene receptor signaling that is mediated by ctr as well as the alternative pathway was prevented. in contrast, the ctr1- protein does not dock at the ethylene receptors and the receptor signaling can be mediated by the alternative, ctr1-independent pathway; thus, ecr2- had little effect on ctr1- phenotype. in line with these results, our data showing that ecr2- prevented etr1- and etr1- but not etr1-11- receptor signaling in ctr1- also support that ecr is involved in the ethylene receptor signaling that is dependent on ctr an alternative explanation for the distinct effects of ecr2- on ctr1- and ctr1- could arise from a role of ecr in ctr stability. in this scenario, very small amount of the ctr1- protein that could associate with the ethylene receptors could have a higher turnover rate in the absence of ecr2; meanwhile, ctr1- reserved in the soluble fraction could be continuously recruited to the receptors and mediate the receptor signaling. contrarily, in ctr1- ctr level is highly reduced without ecr and the receptor signaling is prevented. however, this scenario is not consistent with the argument that the ethylene receptor signaling can be mediated to an alternative pathway that is independent of ctr in other words, if ecr were involved in ctr stability, the ethylene receptor signaling that was mediated independent of ctr still occurred and the ecr2- allele would have little effect on the degree of ctr1- ethylene responses.  ctr is presumably activated by the ethylene receptors, and the mechanism is unknown. little is known about the presence of any other components that are involved in the ethylene receptor signaling to ctr the present data suggest that ecr is a component involved in part in ctr1-mediated ethylene receptor signaling, which indicates a regulatory mechanism for the receptor signaling. we favor the argument that ctr activity but not level was affected by ecr2- in ctr1- with limited sequence information for the 598-kb region that contains ecr the cloning of ecr is currently hampered. complementation of ecr2- by a large dna fragment that contains ecr is ongoing. however, with repeated sequences, transposons, retrotransposons, and sequence gaps in this region, cloning of a large dna fragment is still highly challenging. nevertheless, the cloning of ecr will advance our knowledge of ethylene signaling that involves ctr activation. 
the physical interaction between the ethylene receptor histidine kinase domain and ctr n terminus is essential to the ctr1-mediated receptor signal output. to advance our knowledge of the involvement of ctr1-mediated ethylene receptor signaling, we performed a genetic screen for mutations that enhanced the constitutive ethylene response in the weak ctr1- allele. we isolated a loss-of-function allele of enhancing ctr1- ethylene response and found that ecr2- ctr1- and the strong allele ctr1- conferred a similar, typical constitutive ethylene response phenotype. genetic analyses and transformation studies suggested that ecr acts downstream of the ethylene receptors and upstream of the transcription factors ethylene insensitive and ein3-like, which direct the expression of ethylene response genes. signal output by the n terminus of the ethylene receptor ethylene response can be mediated by a pathway independent of ctr expression of the n terminus of the ethylene-insensitive etr1- but not the full-length isoform rescued the ecr2- ctr1- phenotype, which indicates the involvement of ecr in ctr1-mediated but not -independent, ethylene receptor signaling. ecr is a novel allele involved in the ethylene receptor signaling that is mediated by ctr ctr activation by ethylene receptors may require ecr for suppressing the ethylene response. the signal output of ethylene receptor family members is mediated by unknown mechanisms to activate the raf-like protein constitutive triple response in negatively regulating ethylene signaling.
 the signal output of ethylene receptor family members is mediated by unknown mechanisms to activate the raf-like protein constitutive triple response in negatively regulating ethylene signaling. the physical interaction between the ethylene receptor histidine kinase domain and ctr n terminus is essential to the ctr1-mediated receptor signal output. to advance our knowledge of the involvement of ctr1-mediated ethylene receptor signaling, we performed a genetic screen for mutations that enhanced the constitutive ethylene response in the weak ctr1- allele.  we isolated a loss-of-function allele of enhancing ctr1- ethylene response and found that ecr2- ctr1- and the strong allele ctr1- conferred a similar, typical constitutive ethylene response phenotype. genetic analyses and transformation studies suggested that ecr acts downstream of the ethylene receptors and upstream of the transcription factors ethylene insensitive and ein3-like, which direct the expression of ethylene response genes. signal output by the n terminus of the ethylene receptor ethylene response can be mediated by a pathway independent of ctr expression of the n terminus of the ethylene-insensitive etr1- but not the full-length isoform rescued the ecr2- ctr1- phenotype, which indicates the involvement of ecr in ctr1-mediated but not -independent, ethylene receptor signaling. ecr was mapped to the centromere region on chromosome with incomplete sequence and annotation information and rare chromosome recombination events in this region, the cloning of ecr is challenging and still in progress.  ecr is a novel allele involved in the ethylene receptor signaling that is mediated by ctr ctr activation by ethylene receptors may require ecr for suppressing the ethylene response. arabidopsisethylene signalingctr1etr1ecr2 