Download Ethylene hormone receptor action in Arabidopsis

Review articles
Ethylene hormone receptor
action in Arabidopsis
Caren Chang* and Ruth Stadler
Summary
Small gaseous molecules play important roles in biological signaling in both animal and plant physiology. The
hydrocarbon gas ethylene has long been known to
regulate diverse aspects of plant growth and development, including fruit ripening, leaf senescence and flower
abscission. Recent progress has been made toward
identifying components involved in ethylene signal transduction in the plant Arabidopsis thaliana. Ethylene is
perceived by five receptors that have similarity to twocomponent signaling proteins. The hydrophobic aminoterminus of the receptors binds ethylene, and mutations
in this domain both prevent ethylene binding and confer
ethylene insensitivity to the plant; the carboxyl-terminal
portion of the receptors has similarity to bacterial histidine protein kinases. Genetic data suggest a model in
which ethylene binding inhibits receptor signaling, yet
precisely how these receptors function is unclear. Two
of the receptors have been found to associate with a
negative regulator of ethylene responses called CTR1,
which appears to be a mitogen-activated protein kinase
(MAPK) kinase kinase. BioEssays 23:619±627, 2001.
ß 2001 John Wiley & Sons, Inc.
Department of Cell Biology and Molecular Genetics, University of
Maryland.
Funding agencies: NRI Competitive Grants Program/USDA
(Grant No:98-35304-67-95), Department of Energy (Grant No
02-99ER20329), a German Science Society Fellowship to R. Stadler
and the Maryland Agricultural Experiment Station.
*Correspondence to: Dr. Caren Chang, Department of Cell Biology
and Molecular Genetics, HJ Patterson Hall, University of Maryland,
College Park, MD 20782 USA. E-mail: [email protected]
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AIN,
ACC-INSENSITIVE; CTR, CONSTITUTIVE TRIPLE RESPONSE;
EIL, EIN3-LIKE; EIN, ETHYLENE INSENSITIVE; ERE, ETHYLENE
RESPONSE ELEMENT; EREBP, ETHYLENE RESPONSE
ELEMENT-BINDING PROTEIN; ERF, ETHYLENE RESPONSE
FACTOR; ERS, ETHYLENE RESPONSE SENSOR; ETR, ETHYLENE RESISTANT (now considered as ETHYLENE RECEPTOR);
GAF, cGMP-specific and -stimulated phosphodiesterases, Anabaena
adenylate cyclases and Escherichia coli FhlA; HPK, histidine protein
kinase; HPt, histidine-containing phosphotransfer; MAPK, mitogenactivated protein kinase; RAN, RESPONSIVE TO ANTAGONIST.
BioEssays 23:619±627, ß 2001 John Wiley & Sons, Inc.
Introduction
Both plants and animals utilize hormones to communicate between cells and organs. These messenger molecules enable
the organism to develop and respond to internal and external
cues in a coordinated manner. Interestingly, some signal
molecules are gaseous-nitric oxide is the leading example in
mammals. In plants, the premier example of a gaseous signal
molecule is the simple hydrocarbon ethylene (C2H4). Considered one of the five classic plant hormones, ethylene has
long been known to regulate a diverse array of physiological
and developmental processes. The best known effect of
ethylene is the promotion of fruit ripening.(1) Other notable
processes that are regulated by ethylene include seed
germination, senescence, and responses to stress factors
such as flooding, wounding or pathogen attack.(1)
The molecular basis of plant hormone signaling is a
complex topic that has only begun to be unraveled. Insight
into the ethylene-response pathway has been mainly provided
by molecular genetic studies in Arabidopsis thaliana. Dissection of the ethylene-response pathway began with the isolation
of ethylene-response mutants, using genetic screens that are
based on the ``triple response'' of ethylene-treated seedlings.(2,3) The triple response is a striking morphology adopted
by germinating seedlings exposed to ethylene in the dark. In
Arabidopsis, the triple response consists of shortening and
thickening of the hypocotyl and root, exaggeration of the apical
hook curvature and proliferation of root hairs (Fig. 1) .(2,3) This
altered morphology is thought to facilitate seedling emergence from the soil,(4) as ethylene biosynthesis is triggered by
mechanical stress encountered during germination.(5) In the
laboratory, the triple response can be induced in germinating
seedlings by treatment with either ethylene gas or its chemical precursor 1-aminocyclopropane-1-carboxylic acid (ACC)
(which is taken up by the seedlings and converted to ethylene
by ACC oxidase). Due to the simplicity of the assay, nearly all
of the ethylene-response mutants known today were identified
on the basis of triple response defects. ethylene-resistant
(etr ), ethylene-insensitive (ein) and ACC-insensitive (ain)
mutants show little or no triple response in the presence of high
levels of ethylene or ACC(6,7) (Fig. 1). Constitutive response
mutants (ctr) display the triple response even in the absence
of ethylene (Fig. 1). The order of action for these loci has been
established by epistasis analysis, and many of the corresponding genes have been cloned.(6,7) This has uncovered the
BioEssays 23.7
619
Review articles
Figure 1. Arabidopsis seedlings grown in the dark in the
absence (left) or presence (right) of 10 ml per liter ethylene.
The wild-type (WT) develops the typical triple response
phenotype in the presence of ethylene: a short and thick
hypocotyl with an exaggerated apical hook, and a short and
thick root. The constitutive ethylene-response mutant ctr1-1
develops the triple response phenotype even in the absence
of ethylene, whereas the ethylene-insensitive mutant etr1-1
shows no triple response even in the presence of ethylene.
framework of the ethylene-response pathway; the pathway
starts with ethylene perception by a family of receptors and
progresses to the regulation of ethylene-response genes
(Fig. 2). Recent biochemical and genetic studies have defined
the pathway further. This review will focus on the ethylene
receptors, highlighting findings concerning ethylene receptor
action, their unique properties and some of the questions
currently under investigation.
Overview of the ethylene-response pathway
In Arabidopsis, the molecular cloning of genes involved
in ethylene signaling has revealed a variety of signaling
modules in the ethylene-response pathway (Fig. 2). The first
Arabidopsis ethylene-response mutant that was described
was etr1-1.(2) The ETR1 gene(8) was found to encode a protein
having striking similarity to receptor histidine kinases of the
two-component signaling system.(9) Prior to this, it had been
thought that the two-component signaling mechanism was
exclusive to bacteria. Since then, a number of two-component
homologs have been uncovered in eukaryotes.(10±12) Most
recently, it was discovered that a two-component histidine
kinase in Arabidopsis is the receptor for the plant hormone
cytokinin.(13) Another histidine kinase in Arabidopsis appears
to function as an osmosensor.(14) Two-component pathways
also control osmoregulation and stress responses in yeast,
620
BioEssays 23.7
Figure 2. Schematic diagram of the ethylene signal
transduction pathway in Arabidopsis. Five ethylene receptors
(ETR1, ERS1, ETR2, EIN4 and ERS2) relay the ethylene
signal to the CTR1 protein kinase, which is a negative
regulator of ethylene responses. CTR1 has similarity to the
Raf family of MAPKKKs, and is therefore presumed to be the
first component of a MAP kinase module (which is indicated
by ``?''). EIN2, which acts downstream of CTR1, has an
integral membrane domain with similarity to the Nramp family
of metal-ion transporters. The specific membrane locations of
the ethylene receptors and EIN2 are currently unknown.
Downstream of EIN2, a novel transcription factor called EIN3
promotes transcription of ERF1, which encodes a member of
the EREBP family of transcription factors. ERF1, in turn, binds
to the GCC-box promoter element to activate transcription of
specific ethylene-response genes. With the exception of
EIN3's activation of ERF1 transcription, the solid arrows in
the diagram represent undefined steps in the pathway.
and are involved in Dictyostelium and Neurospora development,(12) but have not been found in animal systems.
The ETR1 protein was suspected to be an ethylene
receptor based on initial genetic and physiological data.(2,8)
The subsequent demonstration that ETR1 reversibly binds
Review articles
ethylene provided strong evidence that ETR1 is indeed an
ethylene receptor.(15) Currently, ethylene perception in Arabidopsis is thought to involve not only ETR1, but four ETR1related genes (ERS1, ETR2, EIN4 and ERS2).(16±18) In recent
years, substantial progress has been made concerning the
structure and function of the ethylene receptors. Diverse
plants such as tomato, muskmelon and carnation contain
similar families of ethylene receptor genes, suggesting that the
mechanism of ethylene signaling is conserved in flowering
plants.(10)
Downstream of the receptors, the next known component is
the protein kinase CTR1. Loss-of-function ctr1 mutants exhibit
pleiotropic constitutive ethylene responses, and thus CTR1 is
thought to be a negative regulator of ethylene responses.(19)
The CTR1 protein kinase domain has high sequence similarity
to the Raf family of mitogen-activated protein kinase kinase
kinases (MAPKKKs), suggesting that CTR1 may act in a
MAPK phosphorylation cascade. In animals, MAPKKKs are
serine/threonine protein kinases, which are activated by
growth factors and mitotic signals. MAPKKKs phosphorylate
MAPKKs, which in turn phosphorylate MAPKs, which regulate
transcription factors. A number of MAPKKK and MAPKK
genes have been isolated in plants; however, it is not known
whether any of these are involved in ethylene signaling.(20)
Regulation of a MAPKKK by two-component histidine kinases
appears to be rare; no animal MAP kinase cascades are
known to be regulated by two-component proteins, and only
two pathways (both in yeast) have been found to contain both a
two-component system and a MAP kinase cascade.(21±23)
EIN2, which acts downstream of CTR1, is one of the central
players in the ethylene-response pathway. Loss-of-function
ein2 mutants are highly ethylene-insensitive for all known
ethylene responses,(3) and also appear to be defective in
their response to several other hormones.(24) The function
of the large EIN2 protein is difficult to predict. The aminoterminal portion of EIN2 consists of twelve putative membrane-spanning regions with similarity to the Nramp family of
metal ion carriers.(24) Ion transport activity, however, has not
been detected for EIN2, and the subcellular localization of
EIN2 is unknown. The novel carboxyl-terminal portion is
predicted to be a coiled coil, suggesting a role in proteinprotein interactions. Overexpression of truncated or wild-type
EIN2 in Arabidopsis indicates that EIN2 is an ethyleneresponse activator with an autoinhibitory amino-terminal
domain.(24)
A common destination of cellular signaling pathways is
the nucleus, where specific transcription factors control gene
expression. Two types of ethylene-responsive transcriptional
regulators have been identified: the family of ETHYLENE
INSENSITIVE3 (EIN3) (and EIN3-LIKE (EIL) proteins)(25,26)
and the family of ETHYLENE RESPONSE ELEMENTBINDING PROTEINS (EREBPs).(27±29) Several members of
the EIN3/EIL family have been shown to bind a conserved cis-
element in the promoter of an EREBP-encoding gene called
ETHYLENE RESPONSE FACTOR1 (ERF1).(26) EIN3 activates transcription of ERF1, and ERF1 in turn binds to a cisacting sequence known as the GCC-box located in the
promoters of secondary target genes, such as those encoding
basic chitinase and defensin PDF1.2.(26,30) ERF1 is thought
to be a transcriptional activator, since overexpression of ERF1
activates many ethylene responses. Thus, a signal amplification cascade exists at the transcriptional level to activate
ethylene-response genes (Fig. 2).(6)
Structurally diverse ``two-component''
ethylene receptors
The two-component system is a signaling mechanism used
primarily by bacteria to respond to a broad array of environmental signals.(9) Escherichia coli alone has at least 30
different pairs of sensor/response regulator proteins.(9) Sensor kinases and response regulators are the two principal
players of the two-component signaling cascade (Fig. 3) .(9)
The sensor kinase (receptor-like) component typically comprises two domains: an amino-terminal input domain, which
perceives the signal, and a carboxyl-terminal histidine protein
kinase (HPK) domain, which transmits the signal. Sequence
comparison of several hundred sensor kinases have indicated
that the HPK domains are conserved, whereas the input
domains appear to be unrelated modules. The prototypical
response regulator consists of a conserved receiver module
and a variable output domain that mediates downstream
responses. Two-component pathways share the following
His-Asp signaling mechanism: signal perception regulates
autophosphorylation of a conserved His residue in the sensor
kinase, and the phosphoryl group is then transferred to a
conserved Asp residue in the receiver domain of the response
regulator, thereby modulating the activity of the output domain
(Fig. 3). Known output domains include transcription factors or
regulators of kinase activities. Numerous configurations of
this basic two-step pathway have been identified.(9) In some
cases, as in the ethylene receptors, the response regulator
is not an independent polypeptide but a carboxyl-terminal
domain of the sensor, in which case the receptor is called a
hybrid HPK.(9,31) Some hybrid HPKs transmit the signal via a
multistep phosphorelay involving a third component known as
a histidine-containing phosphotransfer (HPt) domain protein.
The HPt protein serves as a phosphohistidine intermediate in
the transfer of the phosphoryl group from a receiver domain
onto another receiver, resulting in a His-Asp-His-Asp phosphorelay (Fig. 3).(9,31)
The ethylene receptor family has sequence similarity to
both the sensor kinase and hybrid HPK of the two-component
system (Fig. 4). In Arabidopsis, all five ethylene receptors
(ETR1, ERS1, ETR2, EIN4, ERS2) have an amino-terminal
ethylene-binding domain (described in the next section), and
the carboxyl-terminal portion has similarity to HPKs. Three of
BioEssays 23.7
621
Review articles
Figure 3. Basic features of the two-component signaling system. Signal perception by the input domain of the sensor kinase regulates
histidine protein kinase activity. Following autophosphorylation of a conserved histidine residue, the phosphate is transferred to a
conserved aspartate residue that is either in the receiver domain of the cognate response regulator (lower solid arrow) or, in the case of
a hybrid HPK, in a covalently-attached receiver domain (dashed arrow). In the former situation, the receiver domain directly modulates
the output domain, which is the effector for downstream responses. In the His-Asp-His-Asp multistep phosphorelay (dashed arrows),
there is a histidine-containing phosphotransfer (HPt) domain protein, which serves as a phosphohistidine intermediate in the transfer of
the phosphoryl group from the receiver of the hybrid HPK onto the receiver of a response regulator.
the receptors (ETR1, ETR2 and EIN4) also have a covalently
attached carboxyl-terminal receiver domain, and are therefore
considered to be hybrid HPKs (Fig. 4). ERS1 and ERS2 lack a
receiver domain, suggesting that these receptors may have a
distinct signaling circuit, or perhaps they signal to a receiver
Figure 4. Structure of Arabidopsis ethylene receptor family.
The ethylene-binding domain is located in the membrane
anchor of the receptors, which comprises either three (solid
vertical bars) or four hydrophobic regions. (The fourth region
is designated by a shaded bar.) A GAF-related domain of
unknown function lies in the presumed cytoplasmic portion,
next to the ethylene-binding domain.(63) The table indicates
whether the domains are present (Y) or absent (N) for each
receptor. All five receptors contain a histidine protein kinase
domain, but only ETR1 and ERS1 possess the conserved
motifs (N, G1, F and G2)(9) that are considered necessary for
histidine kinase activity. ETR1, ETR2 and EIN4 are structurally similar to hybrid histidine protein kinases, since they
have a covalently attached carboxyl-terminal receiver domain.
622
BioEssays 23.7
domain of one of the hybrid HPK receptors. To date, the only
demonstrated activity (other than ethylene binding) for any of
the receptors is in vitro autophosphorylation of ETR1 on the
conserved histidine residue.(32)
If the ethylene receptors function as prototypical twocomponent signaling proteins, then each of the hybrid HPK
receptors should act in a multistep phosphorelay employing an
HPt protein followed by a response regulator protein, whereas
the non-hybrid receptors would each be paired with a separate
response regulator. Several functional HPt homologs,(11,33,34)
and over a dozen plant response regulator homologs have
been identified,(35) and the signaling roles of these proteins are
currently being investigated. Several Arabidopsis HPt homologs can interact with ETR1 in the yeast two-hybrid assay.(36)
As presented in the remainder of this article, however, the
actual signaling mechanism may be more complex than a twocomponent phosphorelay. For example, ETR1 and ERS1
contain all of the conserved sequence motifs that characterize functional histidine kinases, yet the other three receptors actually lack most or all of these motifs, raising the
question of whether the latter three possess histidine kinase
activity (Fig. 4). The expected target of two-component
signaling, namely an output domain regulated by a receiver
domain, is also unknown. Thus, the role of phosphorylation
and the extent of similarity to ``classic'' two-component
signaling pathways have yet to be established for ethylene
signaling.
A sequence of unknown function, which has similarity to
GAF domains, is situated between the ethylene-binding
domain and the HPK domain. GAF domains are found in
diverse phototransducing proteins in eukaryotes and prokaryotes, including the plant photoreceptor phytochrome. In
some proteins, the GAF domain is involved in binding of
cGMP.(37) Since phytochrome is believed to have evolved
from prokaryotic two-component histidine kinase photorecep-
Review articles
tors,(38,39) the ethylene receptor may have arisen from an
ancestral prokaryotic photoreceptor upon the addition of an
ethylene-binding domain. The genome of the cyanobacteria
Synechocystis in fact encodes a sequence that aligns with the
input domain of the ethylene receptors, and the protein product
is capable of binding ethylene.(40) The encoded protein has no
sequence similarity to two-component proteins, however, and
its role in Synechocystis is unknown.
Ethylene binding
Several lines of evidence suggest that ETR1 and its homologs
function as receptors for ethylene. Dominant mutations in all
five ethylene receptor genes confer pleiotropic ethylene
insensitivity,(2,16,18) and examination of etr1-1 mutant plants
revealed a reduced ability to bind ethylene.(2) The most compelling argument that these proteins are receptors is that ETR1
and ERS1 reversibly and saturably bind ethylene, as shown
using a yeast cell expression system.(15,40,41) The binding
occurs with a dissociation constant of 0.04 ml/L gaseous ethylene and a half-life of 12 hours, both of which are consistent
with rates observed in ethylene-binding/response assays
in plants.(42±44) The ethylene-binding domain of ETR1 lies
within the first 128 amino acids ,(15,40) and this domain is
the most conserved region among the five Arabidopsis
ethylene receptors (44±54% amino acid identity). In the yeast
expression system, mutations in this domain can result in the
loss of ethylene-binding ability.(15,40,41,45) The binding site
appears to consist of three hydrophobic regions, which
perhaps serve as a membrane anchor.(40,46) ETR1 indeed
localizes to plant membrane fractions,(24) however the identity
of the particular membrane is still under investigation.
Since ethylene is 14-fold more soluble in lipid than in water,
a membrane-localized binding site for ethylene is not
unreasonable.(1)
A transition metal was predicted to be necessary for
ethylene binding,(48) and consistent with this hypothesis,
ETR1 was shown to bind ethylene only in the presence of
copper ions.(40,45) Interestingly, a gene involved in the proper
functioning of the ethylene receptors, RAN1, codes for a
copper transporting P-type ATPase.(49,50) ran1 mutants develop a constitutive ethylene-response phenotype similar to that
of multiple receptor loss-of-function mutants (which are
discussed later). The RAN1 protein is located in intracellular
membrane compartments and is believed to deliver copper
ions from storage sources inside the cells. ran1 loss-offunction mutants display a rosette lethal phenotype, which is
thought to be caused either by general effects due to the
reduced copper availability for other copper-utilizing enzymes,
or by disruption of ethylene receptor function.(49,50) Notably,
plants containing loss-of-function mutations in four of the five
ethylene receptor genes (all but ERS1) also often died before
flowering.(51)
Inhibition of receptor signaling by ethylene
An interesting feature of the ethylene receptors is that they are
negative regulators of the ethylene-response pathway,
i.e. they repress ethylene responses in the absence of the
ethylene signal (Fig. 5). This is counterintuitive to the notion of
receptors being activated by their signals to turn on responses.
The negative regulation by the ethylene receptors was
revealed through the analysis of severe loss-of-function or
null mutants, which were isolated for four of the Arabidopsis
ethylene receptor genes.(51) (Loss-of-function mutants for
ETR1, ETR2 and EIN4 were identified among revertants of
dominant ethylene-insensitive mutants for these genes, and a
loss-of-function ERS2 allele was obtained by a T-DNA
insertion. An ERS1 loss-of-function mutant has not yet been
described.) Single ethylene receptor mutants showed essentially wild-type phenotypes, thus indicating that the genes have
redundancy. In contrast, when the receptor mutants were
crossed together, the resulting homozygous triple and quadruple loss-of-function mutants displayed constitutive ethylene
responses. Since the absence of receptors resulted in
constitutive responses, the wild-type receptors must be active
repressors (i.e. negative regulators) of ethylene responses.
This suggests a model in which in the receptors repress
ethylene responses when ethylene is unbound (Fig. 5).
Consequently, when ethylene is bound, the receptors are
inactivated leading to ethylene responses.(51,52)
These results provide a probable explanation for why only
dominant ethylene receptor mutants have been isolated in
Figure 5. Model for ethylene receptor action. The receptors
are negative regulators of the ethylene signaling pathway,
since multiple receptor loss-of-function mutants develop a
constitutive ethylene-response phenotype. When ethylene is
not bound (A), the receptors interact and activate CTR1,
which results in the inhibition of ethylene responses. When
ethylene is bound (B), the receptors are inactive with respect
to activating the downstream CTR1 protein. The ethylene
insensitivity of the dominant receptor mutants is caused by a
receptor protein that is constitutively active due, in some
cases, to the inability to bind ethylene.
BioEssays 23.7
623
Review articles
mutant screens. Any single loss-of-function receptor mutants
would be wild-type in appearance, whereas dominant receptor
mutants could be isolated by virtue of their ethyleneinsensitive phenotype. Since the dominant mutant phenotype
is opposite that of the triple and quadruple loss-of-function
mutants, it follows that the dominant receptor mutants are
caused by gain-of function mutations. (Note that the gain of
function is with respect to signaling activity, not with respect to
ethylene binding.)
A basis for the dominance of the
ethylene-insensitive receptor mutations
Based on the conclusion that ethylene receptor signaling is
active in the absence of ethylene binding and inactive when
ethylene is bound, we can attempt to derive a model for how
the dominant mutations confer insensitivity to ethylene. Each
of the dominant ethylene receptor mutations results in an
amino acid substitution in the ethylene-binding domain, and
several of these mutations correlate with a dramatic reduction
in ethylene binding.(45) Conceivably, the inability to bind
ethylene locks the signaling domain (cytoplasmic portion) into
a perpetually active signaling state, such that responses are
constitutively repressed despite the presence of redundant
wild-type family members. Thus, the mutation would be
dominant rather than recessive. This explanation, however,
is unlikely to be the entire basis for the ethylene-insensitive
phenotype. First, the repression activity would have to be very
strong, because several of the dominant mutants are
completely insensitive to ethylene, even when treated with
extremely high doses of ethylene. Second, the fact that we see
constitutive ethylene responses in the quadruple or triple lossof-function mutants (which still carry one or two wild-type
receptor genes, respectively) suggests that having one or two
receptors is insufficient to repress responses. In other words,
the dominant ers1-1 mutant (which carries four redundant
wild-type receptor genes) strongly inhibits the ethyleneresponse pathway,(16) yet the single wild-type ERS1 gene
cannot prevent the constitutive ethylene response in the
quadruple receptor mutant.(51) Therefore, it has been proposed that the mutant gain-of-function receptors are somehow
hyperactive.(46,51,52)
We speculate that hyperactive signaling by the dominant
mutant receptors might be achieved via higher order complexes of ethylene receptors, such that the locked signaling
conformation of a mutant receptor dictates the conformation of
the associated receptors. The complexes might comprise
either multiple receptor homodimers, heterodimers or higher
multimers. There is no evidence yet for or against heterodimer
formation, although ETR1 antibodies detect the ETR1 dimer at
a single specific molecular weight in Arabidopsis membrane
extracts.(47) Evidence from yeast two-hybrid experiments
suggest that the GAF domains of different ethylene receptors
(e.g., ETR2 and ETR1) can associate with each other
624
BioEssays 23.7
(C.K. Wen and C. Chang, unpublished). The capacity for
dimerization is also suggested by the three dimensional
structure of the ETR1 receiver domain.(53) Bacterial histidine
kinases have been shown to form heterodimers and catalyze
transphosphorylation.(54,55) Conceivably, a dominant mutant
ethylene receptor might catalyze transphosphorylation of wildtype ethylene receptors, which would otherwise be inactive.
Why are there multiple ethylene receptors?
The genetic data in Arabidopsis indicate that the receptor
genes have functional redundancy with respect to the seedling
triple response. Single null mutants appear to be wild type,
while the dominant receptor mutations result in a similar
phenotype independent of which receptor gene is mutated.
The expression patterns of the receptor genes also overlap
widely.(17) Families of ethylene receptor genes are found not
only in Arabidopsis, but in other plants as well. So, what might
be the advantage for having multiple receptors? The fact that
ethylene sensitivity varies between tissues and for different
responses suggests a rationale for having multiple receptors.(44) As suggested by Hua and Meyerowitz,(51) the
receptors may differ in their affinity for ethylene, enabling the
plant to respond to a broad spectrum of ethylene concentrations. In addition, the expression patterns of ethylene receptor
genes in tomato implicate tissue-specific roles for the different
genes.(56,57) For example, an ERS1 homolog is highly
upregulated during tomato fruit ripening.(56) Expression of
three of the Arabidopsis ethylene receptor genes are also
induced by ethylene.(17) The upregulation of receptors in
response to ethylene might provide a mechanism for adaptation to ethylene; since the half-life of ethylene binding is
very long, an increase in the number of unbound receptors
may enable the plant to react to changes in ethylene
concentration.
Linking the ethylene receptors to CTR1,
a MAPKKK
The next known component downstream of the ethylene
receptors is CTR1, which is thought to function as a MAPKKK.
In animals, MAPKKKs are typically regulated by tyrosinekinase receptors or by G-protein-coupled receptors.(58) There
are only two known examples of pathways that combine a
MAPK pathway with a two-component system. One is the S.
cerevisiae osmosensing pathway(21,59) and the other is the S.
pombe stress-response pathway.(22,23) In the osmosensing
pathway, the hybrid histidine kinase Sln1 transfers a phosphoryl group from the Asp residue in the Sln1 receiver domain
to a His residue in a separate HPt protein called Ypd1, and then
to an Asp in the response regulator protein Ssk1. Ssk1 interacts with and regulates two downstream acting MAPKKKs,
Ssk2 and Ssk22.(21,59)
The ethylene signaling pathway may differ from this yeast
pathway, since a direct interaction has been observed
Review articles
between CTR1 and both ETR1 and ERS1.(60) The CTR1
amino-terminal domain, which is the presumed regulatory
domain, can associate with the histidine kinase domain of
ETR1 and ERS1 as well as the ETR1 receiver domain, as
shown by the yeast two-hybrid assay and by in vitro copurification techniques. It is not yet known whether these components alone are sufficient to transfer the signal from the
receptors to CTR1. In the yeast osmosensing pathway, the
unphosphorylated form of the response regulator SSK1
appears to directly activate the MAPKKK Ssk2.(21,59) For the
ethylene-response pathway, there may be a similar mechanism involving the ETR1 receiver domain and CTR1. Recently,
the three-dimensional structure of the ETR1 receiver domain
was determined.(53) The structure highly resembles the 3D
structure of previously investigated bacterial receivers such
as Escherichia coli Che Y, even though the sequence identity
between ETR1 and CheY is only 17.6%. Interestingly, receiver
domains (including that of ETR1) show striking structural
similarity to Ras proteins,(53) which are involved in activating
Raf kinases (to which CTR1 is similar). Since Ras and
Raf are known to interact, the mechanistic link between
ETR1 and CTR1 could be similar to the signaling between
Ras and Raf.
CTR1 might be activated by dimerization facilitated by
ETR1. As mentioned earlier, evidence suggests that ETR1
forms homodimers through disulfide linkages in the ethylenebinding domain.(47) In addition, the ETR1 receiver domain
forms homodimers both in solution and in the crystal, as
observed for several other bacterial receivers. For some
receivers, dimerization is independent of the phosphorylation
state, whereas in others, dimerization only occurs in the
unphosphorylated state. The interaction interface in the ETR1
receiver dimer shows high structural similarity to the interface
of phosphorylation-dependent dimerizing receivers. Consequently, Mueller-Dieckmann et al.(53) proposed that dimerization of the ETR1 receiver domain occurs in the
unphosphorylated state, and that phosphorylation would
cause monomerization. Adding CTR1 to the picture, we
speculate that ethylene binding causes autophosphorylation
of ETR1, resulting in the inactivation of the CTR1 kinase
(which is a negative regulator of ethylene responses) perhaps
through the loss of dimerization. That is, if CTR1 is associated
with ETR1, then CTR1 might be dimerized and thus activated
in the absence of ethylene, and the monomerization of the
ETR1 receiver domain upon ethylene-induced phosphorylation might cause loss of CTR1 dimerization, thereby inactivating CTR1.
An association between CTR1 and the ethylene receptors
does not rule out the participation of other proteins acting in a
multimeric interaction complex as shown in bacterial chemotaxis.(61) In addition, it is worth noting that ctr1 null mutants
are still capable of responding to ethylene, indicating the
existence of an alternative pathway for ethylene responses.(62)
Thus, a direct regulation of CTR1 does not preclude a
``traditional'' two-component signaling pathway leading from
the receptors.
Conclusions
Great progress has been made in understanding ethylene
perception and signaling through the use of genetic approaches in Arabidopsis. However, much remains to be uncovered concerning the biochemical functions of and interactions
among the different ethylene receptors. It is still unclear what
role phosphorylation plays in the response pathway and how
the presence and absence of ethylene is relayed to the cell by
the different receptors. For example, does binding of ethylene
modulate histidine kinase activity and, if so, in what manner?
Does phosphorylation-dependent receptor dimerization occur? We know that the receptors repress ethylene responses,
however, the mechanism for this repression is unknown.
Perhaps repression does not involve a biochemical activity,
and inactivation of the repression occurs through histidine
kinase activity elicited by ethylene binding. It is also important
to determine the distinct role provided by each member of the
ethylene receptor family. For this, it will be essential to have a
null mutant of ERS1Ðperhaps ERS1 is a positive regulator of
ethylene responsesÐand to describe the phenotype of plants
that completely lack all five receptor genes. The subcellular
location of the receptors has yet to be determined. Differences
in tissue localization may help to reveal different functions
among the five receptors. Another question that should be
addressed is the process of adaptation to ethylene. This may
involve recycling of receptors, protein degradation and/or
synthesis of new receptors. Finally, new components, such as
regulators of receptor function, and substrates for the
receptors have yet to be revealed. Recent advances in reverse
genetics and Arabidopsis genomics should speed the discovery of such components.
Acknowledgments
We thank our colleagues Stephen Mount, Richard Stewart
and Zhongchi Liu for discussion and comments on the
manuscript.
References
1. Abeles FB, Morgan PW, Salveit ME. ed; Ethylene in Plant Biology. San
Diego. Academic Press. 1992.
2. Bleecker AB, Estelle MA, Somerville C, Kende H. Insensitivity to ethylene
conferred by a dominant mutation in Arabidopsis thaliana. Science
1988;241:1086±1089.
3. GuzmaÂn P, Ecker JR. Exploiting the triple response of Arabidopsis to
identify ethylene-related mutants. Plant Cell 1990;2:513±523.
4. Harpham NVJ, Berry AW, Knee EM, Roveda-Hoyos G, Raskin I, Sanders
IO, Smith AR, Wood CK, Hall MA. The effect of ethylene on the growth
and development of wild-type and mutant Arabidopsis thaliana (L.)
Heynh. Ann Bot 1991;68:55±61.
5. Goeschl JD, Rappaport L, Pratt HK. Ethylene as a factor regulating the
growth of pea epicotyls subjected to physical stress. Plant Phys 1966;41:
877±884.
BioEssays 23.7
625
Review articles
6. Stepanova AN, Ecker JR. Ethylene signaling: from mutants to molecules.
Curr Opin Plant Biol 2000;3:353±360.
7. Johnson PR, Ecker JR. The ethylene gas signal transduction pathway: A
molecular perspective. Annu Rev Genet 1998;32:227±254.
8. Chang C, Kwok SF, Bleecker AB, Meyerowitz EM. Arabidopsis ethyleneresponse gene ETR1: similarity of product to two-component regulators.
Science 1993;262:539±544.
9. Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem 2000;69:183±215.
10. Urao T, Yamaguchi-Shinozaki K, Shinozaki K. Two-component systems
in plant signal transduction. Trends Plant Sci 2000;5:67±74.
11. D'Agostino BD, Kieber JJ. Phosphorelay signal transduction: The emerging family of plant response regulators. Trends Biochem Sci 1999;24:
452±456.
12. Loomis WF, Shaulsky G, Wang N. Histidine kinases in signal transduction
pathways of eucaryotes. J Cell Sci 1997;110:1141±1145.
13. Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata
S, Shinozaki K, Kakimoto T. Identification of CRE1 as a cytokinin receptor
from Arabidopsis. Nature 2001;409:1060±1063.
14. Urao T, Yakubov B, Satoh R, Yamaguchi-Shinozaki K, Seki M, Hirayama
T, Shinozaki K. A transmembrane hybrid-type histidine kinase in
Arabidopsis functions as an osmosensor. Plant Cell 1999;11:1743±
1754.
15. Schaller GE, Bleecker AB. Ethylene-binding sites generated in yeast
expressing the Arabidopsis ETR1 gene. Science 1995;270: 1809±
1811.
16. Hua J, Chang C, Sun Q, Meyerowitz EM. Ethylene insensitivity conferred
by Arabidopsis ERS1 gene. Science 1995;269:1712±1714.
17. Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR,
Meyerowitz EM. EIN4 and ERS2 are members of the putative ethylene
receptor gene family in ±. Plant Cell 1998;10:1321±1332.
18. Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB,
Meyerowitz EM. ETR2 is an ETR1-like gene involved in ethylene
signaling in Arabidopsis. Proc Natl Acad Sci USA 1998;95:5812±
5817.
19. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR. CTR1, a
negative regulator of the ethylene response pathway in Arabidopsis,
encodes a member of the raf family of protein kinases. Cell 1993;72:
427±441.
20. Mizoguchi T, Ichimura K, Yoshida R, Shinozaki K. MAP kinase cascades
in Arabidopsis: their roles in stress and hormone responses. Results
Probl Cell Differ. 2000;27:29±38.
21. Posas F, Wurgler-Murphy SM, Maeda T, Witten EA, Thai TC, Saito H.
Yeast HOG1 MAP kinase cascade is regulated by a multistep
phosphorelay mechanism in the SLN1-YPD1-SSK1 ``two component''
osmosensor. Cell 1996;86:865±875.
22. Buck V, Quinn J, Pino TS, Martin H, Saldanha J, Makino K, Morgan BA,
Millar JB. Peroxide sensors for the fission yeast stress-activated mitogenactivated protein kinase pathway. Mol Cell Biol 2001;12:407±419.
23. Shieh J-C, Wilkinson MG, Buck W, Morgan BA, Makio K, Millar JBA. The
Mcs4 response regulator coordinately controls the stress-activated
Wak1-Wis1-Sty1 Map kinase pathway and fission yeast cell cycle.
Genes Dev 1997;11:1008±1022.
24. Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR. EIN2, a
bifunctional transducer of ethylene and stress responses in Arabidopsis.
Science 1999;284:2148±2152.
25. Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR.
Activation of the ethylene gas response pathway in Arabidopsis by the
nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell
1997;89:1133±1144.
26. Solano R, Stepanova A, Chao Q, Ecker JR. Nuclear events in ethylene
signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 1998;12:
3703±3714.
27. Ohme-Takagi M, Shinshi H. Structure and expression of a tobacco b-1,3glucanase gene. Plant Mol Biol 1990;15:941±946.
28. Buettner M, Singh KB. Arabidopsis ethylene-responsive element binding
protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein
interacts with an ocs element binding protein. Proc Natl Acad Sci USA
1997;94:5961±5966.
626
BioEssays 23.7
29. Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M. Arabidopsis
ethylene-responsive element binding factors act as transcriptional
activators or repressors of GCC box-mediated gene expression. Plant
Cell 2000;12:393±404.
30. Hao D, Ohme-Takagi M, Sarai A. Unique mode of GCC box recognition
by the DNA-binding domain of ethylene-responsive element-binding
factor (ERF domain) in plant. J Biol Chem 1998;273:26857±26861.
31. Wurgler-Murphy SM, Saito H. Two-component signal transducers and
MAPK cascades. Trends Biochem Sci 1997;22:172±176.
32. Gamble RL, Coonfield ML, Schaller GE. Histidine kinase activity of the
ETR1 ethylene receptor from Arabidopsis. Proc Natl Acad Sci USA
1998;95:7825±7829.
33. Miyata S-I, Urao T, Yamagouchi-Shinozaki K, Shinozaki, K. Characterization of genes for two-component phosphorelay mediators with a single
HPt domain in Arabidopsis. FEBS Lett 1998;437:175±178.
34. Suzuki T, Imamura A, Ueguchi C, Mizuno T. Histidine-containing
phosphotransfer (Hpt) signal transducers implicated in His-to-Asp
phosphorelay in Arabidopsis. Plant Cell Physiol 1998;39:1258±1268.
35. Imamura A, Hanaki N, Nakamura A, Suzuki T, Tanaguchi M, Kiba T,
Ueguchi C, Sugiyama T, Mizuno, T. Compilation and characterization of
Arabidopsis response regulators implicated in His-Asp phosphorelay
signal transduction. Plant Cell Physiol 1999;40:733±742.
36. Urao T, Miyata S, Yamaguchi-Shinozaki K, Shinozaki K. Possible His to
Asp phosphorelay signaling in an Arabidopsis two-component system.
FEBS Lett 2000;478:227±232.
37. Aravind L, Ponting CP. The GAF domain: an evolutionary link between
diverse phototransducing proteins. Trends Biochem 1997;22:458±459.
38. Elich TD, Chory J. Phytochrome: If it looks and smells like a histidine
kinase, is it a histidine kinase? Cell 1997;91:713±716.
39. Pepper AE. Molecular evolution: Old branches on the phytochrome
family tree. Curr Biol 1998;8:R117±R120.
40. Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB. A
copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
Science 1999;283:996±998.
41. Hall AE, Findell JL, Schaller GE, Sisler EC, Bleecker AB. Ethylene
perception by the ERS1 protein in Arabidopsis. Plant Physiol 2000;123:
1449±1458.
42. Sanders IO, Harpham NVJ, Raskin I, Smith AR, Hall MA. Ethylene
binding in wild type and mutant Arabidopsis (L.) Heynh. Ann Bot 1991;
68:97±103.
43. Sisler EC. Ethylene-binding components in plants. In Matoo AK, Shuttle
JC. ed; The Plant Hormone Ethylene. Florida. CRC Press. 1991. 81±99.
44. Chen Q, Bleecker, AB. Analysis of ethylene signal transduction kinetics
associated with seedling-growth response and chitinase induction in
wild-type and mutant Arabidopsis. Plant Physiol 1995;108:597±607.
45. Hall AE, Chen QG, Findell JL, Schaller GE, Bleecker AB. The relationship
between ethylene binding and dominant insensitivity conferred
by mutant forms of the ETR1 ethylene receptor. Plant Physiol 1999;12:
291±300.
46. Bleecker AB, Esch JJ, Hall AE, Rodriguez FI, Binder BM. The ethylenereceptor family from Arabidopsis: structure and function. Phil Trans R
Soc Lond 1998;353:1405±1412.
47. Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB. The
ethylene response mediator ETR1 from Arabidopsis forms a disulfidelinked dimer. J Biol Chem 1995;270:12526±12530.
48. Burg, SP, Burg EA. Molecular requirements for the biological activity of
ethylene. Plant Physiol 1967;42:144±152.
49. Hirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S,
Alonso JM, Dailey WP, Dancis A, Ecker JR. RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required
for ethylene signaling in Arabidopsis. Cell 1999;97:383±393.
50. Woeste KE, Kieber JJ. A strong loss-of-function mutation in RAN1 results
in constitutive activation of the ethylene response pathway as well as a
rosette lethal phenotype. Plant Cell 2000;12:443±455.
51. Hua J, Meyerowitz EM. Ethylene responses are negatively regulated by a
receptor gene family in Arabidopsis. Cell 1998;94:261±271.
52. Bleecker AB. Ethylene perception and signaling: an evolutionary
perspective. Trends Plant Sci 1999;4:269±273.
53. Mueller-Dieckmann H-J, Grantz AA, Kim S-H. The structure of the signal
receiver domain of the Arabidopsis ethylene receptor ETR1. Structure
1999;7:1547±1556.
Review articles
54. Pan SQ, Charles T, Jin S, Wu Z-L, Nester EW. Preformed dimeric
state of the sensor protein virA is involved in plant-Agrobacterium
signal transduction. Proc Natl Acad Sci USA 1993;90:9939 ±
9943.
55. Parkinson JS. Signal transduction schemes of bacteria. Cell 1993;73:
857±871.
56. Lashbrook CC, Tieman DM, Klee HJ. Differential regulation of the tomato
ETR gene family throughout plant development. Plant J 1998;15:243±
252.
57. Tieman DM, Klee HJ. Differential expression of two novel members
of the tomato ethylene-receptor family. Plant Physiol 1999;120:165±
172.
58. Blumer KJ, Johnson GL. Diversity in function and regulation
of MAP kinase pathways. Trends Biochem Sci 1994;19:236±
240.
59. Posas F, Saito H. Activation of the yeast SSK2 MAP kinase kinase kinase
by the SSK1 two-component response regulator. EMBO J 1998;17:
1385±1394.
60. Clark KL, Larsen PB, Wang X, Chang C. Association of the Arabidopsis
CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proc
Natl Acad Sci USA 1998;95:5401±5406.
61. Liu Y, Levit M, Lurz R, Surette MG, Stock JB. Receptor mediated
protein kinase activation and the mechanism of transmembrane
signaling in bacterial chemotaxis. EMBO J 1997;16:7231±7240.
62. Larsen PB, Chang C. The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem. Plant Physiol 2001;125: 1061±
1073.
63. Ho Y-SJ, Burden LM, Hurley JM. Structure of the GAF domain, a
ubiquitous signaling motif and a new class of cyclic GMP receptor.
EMBO J 2000;19:5288±5299.
BioEssays 23.7
627