trends in plant science
Reviews
Two-component systems in plant
signal transduction
Takeshi Urao, Kazuko Yamaguchi-Shinozaki
and Kazuo Shinozaki
In plants, two-component systems play important roles in signal transduction in response to
environmental stimuli and growth regulators. Genetic and biochemical analyses indicate that
sensory hybrid-type histidine kinases, ETR1 and its homologs, function as ethylene receptors and negative regulators in ethylene signaling. Two other hybrid-type histidine
kinases, CKI1 and ATHK1, are implicated in cytokinin signaling and osmosensing processes,
respectively. A data base search of Arabidopsis ESTs and genome sequences has identified
many homologous genes encoding two-component regulators. We discuss the possible
origins and functions of these two-component systems in plants.
A
ll living organisms have diverse and sophisticated signaling strategies to recognize and respond to their environmental conditions. Protein phosphorylation is a key
mechanism for intracellular signal transduction in both eukaryotic
and prokaryotic cells. This process is catalyzed by protein
kinases, and these are classified into three major groups based on
their substrate specificities: serine/threonine kinases, tyrosine
kinases and histidine kinases. Histidine kinases in bacteria play
key roles in sensing and transducing extracellular signals including chemotactic factors, changes in osmolarity, and nutrient
deficiency. This signal transduction system is mediated by
phosphotransfer between two types of signal transducers and is
therefore referred to as the ‘two-component system’1. The twocomponent system has been well defined in bacteria, and, in
1993, the existence of a bacterial-type histidine kinase was reported in yeast and in Arabidopsis2,3. Since then, many genes
encoding two-component regulators have been identified in
plants, yeast and Dictyostelium3,4. However, the existence of twocomponent regulators in animals has not been reported. In this
review, we focus on the two-component regulators and their
function in plants.
A simple His-to-Asp phosphorelay: two components
Typically, the two-component system is composed of a sensory
histidine kinase and a response regulator (Fig. 1). The histidine
kinase contains an N-terminal input domain and a C-terminal
kinase domain with an invariant histidine residue. The response
regulator contains an N-terminal receiver domain with an invariant aspartate residue and a C-terminal output domain. For
example, in E. coli, osmotic responses are controlled by an
EnvZ–OmpR two-component system. The input domain of the
sensory histidine kinase EnvZ somehow detects changes in external osmolarity and modulates intrinsic kinase and phosphatase
activities. High osmolarity promotes autophosphorylation of a
histidine residue within its kinase domain, followed by transfer
of the phosphoryl group to an aspartate residue within the
receiver domain of the cognate response regulator OmpR. By
contrast, low osmolarity promotes dephosphorylation of the
phosphorylated OmpR. The phosphorylation state of OmpR
alters the DNA-binding activity of the output domain to control
the transcription of target genes. Thus, a physical signal (e.g.
an environmental stimulus) can be converted to a biochemical
reaction, termed ‘His-to-Asp phosphorelay’, by two-component
signaling systems.
A multistep His-to-Asp phosphorelay: more than
two components
Such a simple His-to-Asp phosphorelay is not the only two-component system. In some cases, two-component systems include
additional signaling modules or motifs and constitute more
complicated phosphorelay circuits (Fig. 1). This additional signaling domain, known as the histidine-containing phosphotransfer
(HPt) domain, was first uncovered in an E. coli anaerobic sensor
ArcB. For instance, an osmosensory two-component system in
Saccharomyces cerevisiae consists of three signaling molecules,
SLN1, YPD1 and SSK1 (Ref. 3). The histidine kinase SLN1 acts
as a transmembrane osmosensor. SLN1 has both a kinase and a
receiver domain within the same molecule. This type of histidine
kinase is referred to as ‘hybrid histidine kinase’. At normal osmolarity, SLN1 is activated to autophosphorylate a histidine residue
within the kinase domain using ATP as a phosphodonor. The
phosphoryl group is transferred sequentially by a phosphorelay
reaction to an aspartate residue within the receiver domain, and
then to a histidine residue in the intermediary molecule YPD1,
and finally to an aspartate residue within the receiver domain of
the response regulator SSK1. The phosphorylated form of SSK1
is incapable of activating an osmosensing HOG1 MAPK cascade.
By contrast, under conditions of high osmolarity, SLN1 is inactivated, enabling unphosphorylated SSK1 to accumulate, which
leads to the activation of the HOG1 MAPK cascade. A similar signaling system also operates in osmosensing in fission yeast. The
response regulator MCS4 regulates a WAK1-WIS1-STY1 MAPK
pathway in Schizosaccharomyces pombe5. Such a multistep phosphorelay reaction is believed to have the potential advantages of
providing multiple regulatory checkpoints for signal cross-talk or
negative regulation by certain phosphatases. Multistep phosphorelays are known to be involved in anaerobic regulation in E. coli,
in sporulation in Bacillus subtilis, and in virulence control in
Bordetella pertussis. The existence of multistep phosphorelay
reactions in both prokaryotes and eukaryotes suggests that similar
mechanisms might be more widely used.
Link between prokaryotic two-component system
and eukaryotic signal transduction system
The best documented linkage between prokaryotic and eukaryotic
two-component signal transduction mechanisms is for the
osmoregulation systems of yeast. As mentioned already, in
S. cerevisiae, exposure to high osmolarity activates the HOG1
MAPK cascade through the SLN1-YPD1-SSK1 two-component
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(99)01542-3
February 2000, Vol. 5, No. 2
67
trends in plant science
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Phosphorelay
intermediate
Histidine
kinase
Response
regulator
(a)
P
H
P
D
OmpR
EnvZ
(b)
P
H
P
D
SLN1
P
H
YPD1
P
D
SSK1
Trends in Plant Science
Fig. 1. Two types of phosphorelay signaling in two-component
systems. The input domain is depicted in pink, the transmembrane
region in grey, the kinase domain in blue, the receiver domain in
light green, the histidine-containing phosphotransfer domain in
dark green and the output domain in purple. H represents histidine
and D represents aspartate. (a) Single step His-to-Asp phosphorelay. The sensory kinase EnvZ recognizes a high osmolarity condition and autophosphorelates a histidine residue in the kinase
domain. The phosphoryl group is then transferred to an aspartate
residue in the receiver domain of the response regulator OmpR.
The phosphorylated OmpR promotes its DNA-binding activity
and activates the transcription of high osmolarity-responsive
genes. (b) Multistep His-to-Asp phosphorelay. Under conditions
of high osmolarity, the sensory kinase SLN1 autophosphorylates
and blocks the activation of the osmoregulatory HOG1 MAPK
cascade through a multistep phosphorelay between an aspartate
residue in the receiver domain, a histidine residue in the phosphorelay intermediate YPD1, and an aspartate residue in the
receiver domain of the response regulator SSK1. By contrast, high
osmolarity inactivates SLN1, resulting in the accumulation of
unphosphorylated SSK1.
ETR1
ETR2
EIN4
CKI1
ATHK1
ERS1
ERS2
ARR3
H
H
H ATHP1 (AHP2) ARR4
ATHP2 (AHP3) (ATRR1, IBC7)
ATHP3 (AHP1) ARR5
(ATRR2, IBC6)
D
D
ARR6
H
ARR7
ARR8 (ATRR3)
Histidine
ARR9 (ATRR4)
kinases
Phosphorelay
intermediate
D
ARR1
ARR2
ARR10
ARR11
ARR12
ARR13
ARR14
D
Response
regulators
system. At high osmolarity, the unphosphorylated response
regulator SSK1 activates SSK2 and SSK22 (MAPKKK) by direct
protein–protein interaction. The activated SSK2 and SSK22 phosphorylates PBS2 (MAPKK), and HOG1 (MAPK) is activated.
Furthermore, it should be noted that, in addition to SLN1, another
osmosensor, SHO1, is known in budding yeast3. SHO1 is not a
member of the two-component family, but it acts as a transmembrane osmosensor, activating PBS2 MAPKK by direct interaction
between the SH3 domain of SHO1 and the proline-rich region
of PBS2. SLN1 and SHO1 appear to have a different salt concentration dependency and a different time course for providing an
optimal response to osmolarity changes. In yeast, multiple MAPK
cascades are involved in the responses to a variety of external
stimuli. For example, STE11 (MAPKKK) shares two independent
pathways, an osmosensing SHO1–HOG1 pathway and a pheromoneresponse FUS3/KSS1 pathway. Nevertheless,osmotic stress, which
leads to the activation of the HOG1 MAPK cascade, never activates the FUS3/KSS1 MAPK cascade. Thus, the two pathways
respond to a specific signal, and function independently. This
functional separation is achieved by a scaffold protein that prevents
undesirable signals entering and interfering with the pathway. In
any event, it should be emphasized that the prokaryotic-like twocomponent signaling mechanism can be linked downstream to typical eukaryotic (MAPK) signaling cascades, in yeast. This intriguing
view is further supported by recent findings that two-component
systems in Dictyostelium play important roles in signal recognition
and development, as described below.
A gene, dokA, encoding a hybrid histidine kinase has been
cloned from Dictyostelium6. The dokA deletion mutants show a
dramatic reduction in viability after exposure to medium of high
osmolarity and inhibition of growth and development under less
stringent osmolarity conditions. DokA, unlike EnvZ and SLN1,
does not contain extensive hydrophobic regions, suggesting a
soluble intracellular signal transducer. The signals sensed by
DokA might be either concentrated intracellular solutes or secondary molecules, such as polyamines and trehalose derivatives.
Alternatively, DokA might perceive osmolarity changes by indirectly monitoring the medium using a separate sensory protein.
The development of the Dictyostelium fruiting body is controlled
by an RdeA–RegA two-component system7. RegA has two
functional domains, one is homologous to a mammalian cAMP
phosphodiesterase and the other to bacterial response regulators;
subsequent analysis has revealed that phosphorylation of the
RegA receiver domain stimulates the output activity of the phosphodiesterase domain8. RdeA has weak homology to the yeast
phosphorelay intermediate YPD1, which might complement an
rdeA null mutant9. Indeed, direct phosphotransfer from RdeA to
RegA has been demonstrated in vitro, and it therefore appears that
RdeA is an immediate upstream factor for RegA. Although a
histidine kinase that functions together in this signaling pathway
has not been identified, a multistep phosphorelay two-component
system might play a role in controlling the development of the
fruiting body in Dictyostelium. In this case, it is interesting to
emphasize that the RdeA–RegA two-component system appears
to be linked to the eukaryotic PKA (cAMP-dependent protein
kinase) signaling system.
Trends in Plant Science
Fig. 2. Arabidopsis two-component systems. The extracellular
domain is depicted in pink, the transmembrane region in grey, the
kinase domain in blue, the receiver domain in light green, the
histidine-containing phosphotransfer domain in dark green and the
output domain in purple. H represents histidine and D represents
aspartate.
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February 2000, Vol. 5, No. 2
Histidine kinases as ethylene receptors in Arabidopsis
Several histidine kinases have been cloned and shown to be
involved in the perception of plant hormones and environmental
signals. In Arabidopsis, several ethylene response mutants have
been isolated and extensively characterized. The etr1, etr2 and
ein4 mutants have dominant ethylene insensitivity and are supposed to act at an early step in ethylene signal transduction4,10–13.
trends in plant science
Reviews
The ETR1 gene was first isolated by mapbased cloning2. Subsequently, ETR2 and
Cu RAN1
EIN4 were also cloned14,15. Sequence analyETR1
sis has revealed that three genes enERS1
Air
ETR2
CTR1
EIN2
EIN3
OFF
code hybrid histidine kinases, with three
ERS2
hydrophobic transmembrane regions at the
EIN4
N-terminus but no apparent extracellular
domains (Fig. 2). ETR1 exhibits autophosphorylation activity and binds directly
Cu RAN1
to ethylene as a homodimer by means of
ETR1
disulfide bonds between the cysteine residues
ERS1
Ethylene
16–18
ETR2
CTR1
EIN2
EIN3
ON
within the transmembrane region .
ERS2
EIN4
These results, together with the etr1 mutant
Trends in Plant Science
phenotype, provide evidence that ETR1 is
an ethylene receptor. In addition, two
Fig. 3. A current model of ethylene signal transduction. In wild-type Arabidopsis, ETR1 is
in an active state in the absence of ethylene and activates CTR1 directly or indirectly.
ETR1-related genes, ERS1 and ERS2, have
The activated CTR1 represses the ethylene responses by inactivation of the downstream
been cloned from Arabidopsis based on the
components EIN2 and EIN3. Binding ethylene to ETR1 modulates the activity to an inactive
sequence similarity with ETR1, and shown
form. The inactivated ETR1 inactivates CTR1 and consequently permits ethylene responses.
to be involved in ethylene perception14,19.
Thus, ETR1 and CTR1 function as negative regulators in ethylene action. RAN1 participates
Both proteins contain a kinase domain but
in delivering copper ions to functional ethylene receptors. In the etr1 mutant, the gainnot a receiver domain, unlike ETR1, EIN4
of-function mutation results in the constitutive activation of ETR1 and the ethylene response
and ETR2 (Fig. 2). Expression of altered
is repressed by the activated CTR1 even in the presence of ethylene. Therefore, the etr1
ERS1 and ERS2 genes, in which a nucleotide
mutant becomes insensitive to ethylene. By contrast, in the ctr1 mutant, the loss-of-function
corresponding to the etr1-4 mutation was
mutation results in constitutive inactivation of CTR1 and permits ethylene responses even in
changed, conferred dominant ethylene inthe absence of ethylene. Therefore, the ctr1 mutant shows constitutive ethylene responses.
sensitivity to wild-type plants, suggesting
that ERS1 and ERS2 have important roles
in ethylene sensing. Thus, it was proposed
that Arabidopsis has at least five ETR1-related proteins and that they work at a different phase in ethylene perception. In this
they have, at least in part, redundant functions in ethylene signal- regard, it is noteworthy that the RNA levels of ETR1 and EIN4 are
ing. However, it was uncertain whether all five genes actually constant upon ethylene treatment, whereas the ETR2, ERS1 and
encode ethylene receptors, because their recessive mutants (loss- ERS2 genes are up-regulated by ethylene14. Therefore, the upof-function) could not be obtained by the previous genetic regulation of ethylene receptors is likely to be involved in a mechscreens. In fact, ETR2 and ERS2 have unusual structural features: anism for the adaptation to ethylene, because the induction of
the conserved histidine residue, which is crucial for autophospho- these proteins leads to a higher accumulation of active proteins
rylation is replaced by a glutamate or aspartate residue. It was that can reduce the ethylene responses. An Arabidopsis mutant,
therefore possible that these proteins are not functional ethylene responsive-to-antagonist 1 (ran1) shows ethylene-treated phenoreceptors or that they might not be involved in ethylene sensing types in response to a treatment with trans-cyclo-octene, a potent
directly. Moreover, whether these ethylene receptors are positive receptor antagonist21. The gene for RAN1 encodes a protein with
regulators or negative regulators in ethylene signal transduction, similarity to copper-transporting P-type ATPase, human Menkess/
or whether they are functional receptors had not been elucidated. Wilson disease proteins and yeast Ccc2p. RAN1 possesses copperTo address this issue, loss-of-function mutants (knock-out) of four transporting activity. Based on in planta demonstration using transmembers, ETR1, ETR2, EIN4 and ERS2 genes, have been isolated genics, ethylene signaling requires copper as a cofactor and RAN1
and characterized20. The single loss-of-function mutant showed functions to create functional ethylene receptors by delivering
normal ethylene sensitivity, explaining the failure to isolate the copper ions (Fig. 3). Indeed, the addition of a copper ion leads to
recessive mutations in the genetic screens and their functional an increase in the ethylene-binding activity of ETR1 expressed in
redundancy. By contrast, the quadruple loss-of-function mutant of yeast22.
four members showed strong constitutive ethylene responses in
the absence of ethylene. Thus, the loss-of-function mutant of all Histidine kinases as ethylene receptors in other plants
four genes is phenotypically opposite to their dominant mutants, It has been suggested that several histidine kinases, from various
leading to the conclusion that the dominant mutants are because of species of higher plants, are involved in ethylene perception.
gain-of-function mutations and that they are locked in an active Ethylene plays a key role in ripening climacteric fruits, such as
state that represses ethylene responses, regardless of whether eth- tomato and melon. The NR gene has been cloned from a tomato
ylene is present or absent. This conclusion also means that the ethylene-insensitive mutant Never-ripe and shown to be an ERS1
normal proteins function as negative regulators in ethylene action. homolog (i.e. NR has a kinase domain but not a receiver domain23).
According to a model proposed currently (Fig. 3), the ethylene The expression of the NR gene was positively regulated by ethylene
receptors are in an active state (ON) and repress the response and by the developmental program. Moreover, previous RFLP
(OFF) in the absence of ethylene. When ethylene binds to the mapping studies have revealed that tomato contains at least five
receptors, the activity somehow changes to an inactive state distinct chromosomal loci that hybridize to the ETR1 gene as a
(OFF), releasing the repression (ON). Then, the question arises – probe24,25. In addition to NR, LeETR1 and LeETR2, which are
how do five ethylene receptors play individual roles in ethylene highly homologous to Arabidopsis ETR1, have been cloned from
perception? One possible explanation is that each receptor has tomato25–27. The LeETR1 gene is constitutively expressed in all
a different ethylene-binding affinity or that they function as a tissues, whereas the LeETR2 gene is expressed at low levels
heterodimer or a receptor complex. Another possibility is that throughout the plant, but at high levels in imbibing seeds before
February 2000, Vol. 5, No. 2
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germination. More recently, homologs of ETR1 and ERS1, CmETR1 and Cm-ERS1, respectively, have been isolated from
melon28. The level of Cm-ERS1 mRNA increases during early
fruit development and decreases at the end of fruit enlargement,
whereas the level of Cm-ETR1 mRNA is high in the seeds and
placenta of developing and in fully enlarged fruit. During fruit
ripening, the level of Cm-ERS1 mRNA increases slightly,
whereas the level of the Cm-ETR1 mRNA markedly increases in
the pericarp. Thus, the gene expression of the two putative ethylene receptors is differentially regulated with the developmental
stage and in the tissue of mature fruit. Genomic Southern blot
analysis indicated the presence of an additional related gene in
melon. The flooding response is also known to be initiated by ethylene. As the first step in understanding the molecular mechanism
of the flooding responses, RP-ERS1, a homolog of Arabidopsis
ERS1 and tomato NR, has been cloned from Rumex palustris, a
flooding-tolerant plant29. RP-ERS1 gene expression is induced by
flooding, ethylene, carbon dioxide and low oxygen. Some additional homologous genes appear to exist in R. palustris. Thus,
plants, at least climacteric fruits, appear to have two types of
ethylene receptor, an ethylene-inducible type (ERS1-like family)
and a constitutive-expression type (ETR1-like family), which
might lead to distinct ethylene sensitivities.
Histidine kinases involved in cytokinin signaling
Plants appear to use a histidine kinase as a cytokinin receptor. In
Arabidopsis, several mutants (cki1-1, -2, -3, and -4, and cki2) that
show typical cytokinin responses in the absence of exogenous
cytokinin have been isolated by activation tagging30. A gene,
CKI1, has been cloned and shown to encode a novel hybrid histidine kinase (Fig. 2). Transformation of Arabidopsis wild-type
plants with CKI1 under the control of a single CaMV 35S promoter has confirmed that cytokinin-independent growth is caused
by the overproduction of CKI1. Ectopic expression of CKI1 could
lead to the overestimation of low levels of endogenous cytokinin,
which are usually unable to trigger specific responses. A gene for
CKI2 has been isolated and shown to encode a hybrid histidine
kinase (T. Kakimoto, pers. commun.). The structure of CKI2 is
different from that of CKI1. CKI1 and CKI2 might act as cytokinin receptors with distinct signaling mechanisms.
Hybrid histidine kinase functions as an osmosensor
in plants
Recently, the possible involvement of a histidine kinase in
osmosensing in plants has been shown. A hybrid histidine kinase
ATHK1 has been cloned from Arabidopsis31. ATHK1 contains
two hydrophobic transmembrane regions adjacent to a putative
extracellular domain in the N-terminal half, suggesting functional
similarity with the yeast osmosensor SLN1 (Fig. 2). This possibility was demonstrated by analyzing both the sensing (input) and the
catalytic (output) activities of ATHK1 using yeast osmosensingdefective mutants. ATHK1 can suppress the sln1-ts (sln1-4)
mutant. By contrast, the substitution of either putative phosphorylation site, His or Asp, failed to complement the sln1-ts mutant,
indicating that ATHK1 acts as a histidine kinase in yeast and that
ATHK1 is in an active state in the absence of external signals (e.g.
high osmolarity). Moreover, ATHK1 allowed a yeast mutant lacking both osmosensors, SLN1 and SHO1, to activate HOG1 and to
grow normally under conditions of high osmolarity, suggesting
that the ATHK1 activity changed to an inactive state from an
active state in response to the increase in external osmolarity.
Thus, ATHK1 appears to have an ability to sense and transduce a
signal of external osmolarity to the downstream targets in yeast.
The ATHK1 mRNA was more abundant in roots than other tissues
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February 2000, Vol. 5, No. 2
and accumulated under the conditions of high-salinity and low
temperature. The high level of expression in roots suggests that
ATHK1 is necessary for efficient sensing of environmental signals, such as high-salinity and drought. What are the advantages
of the up-regulation of osmosensors? If increased osmotic pressure causes a conformational change in ATHK1 and consequently
its kinase activity changes to an inactive state, the newly produced
ATHK1 under conditions of high-osmolarity would also be inactive in the membrane, leading to the activation of downstream
pathways. Accumulation of inactive ATHK1 appears to be required to enhance the responses or to reduce the responses quickly
by the reactivation of ATHK1 kinase activity when osmotic stress
is relieved.
Response regulators in higher plants
In addition to the histidine kinases mentioned already, many response regulators have also been isolated in Arabidopsis. Recently,
it has been proposed that they could be classified into two groups,
type-A and type-B, based on their architecture32. The type-A response regulators are mainly composed of a receiver domain and
short N- and C-terminal extensions, whereas the type-B response
regulators have a receiver domain and a largely extended C-terminal
region that is supposed to be an output domain (Fig. 2). ARR3-7,
members of the type-A group, were initially cloned in Arabidopsis
using an EST database33. By using in vitro phosphotransfer analysis, ARR3 has been shown to accept a phosphoryl group from a
phospho-HPt domain of an E. coli hybrid, histidine kinase ArcB.
In addition, overexpression of ARR3 results in the reduction of
the OmpC gene expression, suggesting that ARR3 competes with
OmpR, a transcriptional activator for the OmpC gene, by titrating
the phosphoryl group from the HPt domain of ArcB. It is thus demonstrated that ARR3 has a functional receiver domain as a phosphoacceptor. ARR1 and ARR2, members of the type-B group, have
been cloned subsequently and shown to contain a large extended
C-terminal region, a part of which has a weak homology to single
Myb DNA-binding proteins from potato34. Indeed, their C-terminal
regions are rich in glutamate and proline, which is one of the characteristics of eukaryotic transcriptional activation domains. Similarity to the Myb DNA-binding proteins has also been found in
other members of the type-B group32. Although the DNA-binding
activity of the type-B response regulators has not been demonstrated, the conservation of this region might imply the functional
importance of this region in the regulation of biological function,
such as gene expression.
The two classes of response regulators differ not only in their
structural features, but also in their expression patterns and biochemical activities. The genes for the type-A response regulators,
ARR3, ARR4 (ATRR1, IBC7), ARR5 (ATRR2, IBC6), ARR6 and
ARR7, are induced by exogenous cytokinins, but not by any other
plant hormones35,36. Moreover, re-application of nitrate to Nstarved plants also results in the accumulation of their transcripts,
as previously observed in maize ZmRR1 (Ref. 37). These results
suggest that these cytokinin-responsive ARRs are involved, at
least in part, in the nitrate signal transduction mediated by
cytokinin in Arabidopsis. In this regard, it is noteworthy that an
Arabidopsis histidine kinase CKI1 is implicated to function as a
cytokinin receptor. Some of these ARRs might work together with
CKI1 in a certain cytokinin-sensing two-component system. In
contrast with the type-A ARRs, ARR1, ARR2 and ARR10, members of the type-B group, do not respond to these treatments38.
Thus, two types of ARRs show distinct cytokinin- and nitrateresponsiveness. Moreover, the type-A members can be further
classified into two subgroups, based on their responsiveness to
stress treatments. Among the type-A members, the ATRR1 (ARR4,
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IBC7) and ATRR2 (ARR5, IBC6) genes are induced by high salinity and low temperature, whereas the transcript levels of ATRR3
and ATRR4 are not affected by any of the stress treatments that
have been tested39. In in vitro phosphotransfer analysis, ARR3 and
ARR4, members of the type-A group, have been shown to have an
ability to accept a phosphoryl group from the phosphorylated HPt
domain of AHP1 and AHP2, Arabidopsis phosphorelay intermediates32. By contrast, ARR10, a member of the type-B group, is
incapable of exhibiting phospho-acceptor activity toward any of
the AHPs tested.
Phosphorelay intermediates containing a His-containing
phosphotransfer domain in higher plants
In Arabidopsis, a third component has simultaneously been
cloned by two independent groups40,41. Three Arabidopsis phosphorelay intermediates, ATHP1 (AHP2), ATHP2 (AHP3) and
ATHP3 (AHP1), contain an HPt domain with a conserved histidine and some invariant residues that are crucial for phosphorelay
(Fig. 2). These are somewhat similar to the yeast YPD1 in the
sense that they are small polypeptides containing only an HPt
domain. Indeed, functional analysis using a yeast ypd1 mutant has
demonstrated that all ATHPs act as phosphorelay intermediates
between SLN1 and SSK1 in yeast40. Moreover, the recombinant
AHP1 protein could be phosphorylated by an uncertain hybridtype histidine kinase in the E. coli membrane fractions as a phospho-donor41. Subsequent analysis has revealed that the phosphoryl
group on the histidine residue of AHP1 is transferred transiently
to the receiver domain of ARR3 and ARR4 in vitro41. Thus, it has
been shown that AHP1 (ATHP3) exhibits an ability to accept and
transfer the phosphoryl group directly to the response regulators
in Arabidopsis as well as in yeast. Recently, a phosphorelay intermediate, ZmHP2, has been cloned from maize42. In vitro experiments have shown that ZmHP2 is phosphorylated by E. coli inner
membrane vesicles, and that the phosphoryl group on ZmHP2 is
transiently transferred to ZmRR1 and ZmRR2 response regulators
from maize.
to the hypothesis that CTR1 is a part of an ethylene receptor complex and that the activity of CTR1 is regulated by direct interaction with the two-component histidine kinases. However, other
two-component molecules, such as response regulators and phosphorelay intermediates, have also been isolated from Arabidopsis.
Based on the current two-component system scheme, it is reasonable to suppose that other components are also involved in
the formation of the receptor-CTR1 complex, and that CTR1
activity is regulated by output activity initiated by ethylene
receptor-mediated phosphorelays.
Secondly, in vitro phosphotransfer analysis has indicated that
ARR3 and ARR4 have an ability to accept a phosphoryl group
from the phosphorylated HPt domain of AHP1 and AHP2,
whereas ARR10 is incapable of exhibiting a phosphoacceptor
activity toward any AHPs tested32. AHP1 or AHP2 might be an
upstream phosphorelay mediator of ARR3 or ARR4, and they
might function together in a particular two-component system.
Two-hybrid screening has identified two closely related proteins,
ATDBP1 and ATDBP2, as proteins that interact with the response
regulator ARR4 (Ref. 45). ATDBP1 has sequence similarity with
Remorin from potato, which is a membrane-associated and
uranide-binding phosphoprotein. ATDBP1 and ATDBP2 might
be a downstream target or an upstream regulator for ARR4, because such small response regulators lacking a presumed output
domain probably function as an on–off switch molecule at an
intermediate step in His-to-Asp phosphorelay. Alternatively,
ATDBP1 and ATDBP2 might assist ARR4 in the localization to the
plasma membrane where a certain two-component system works.
A phosphorelay network can be assumed by the similarity of
their expression profiles. The ATHP3 gene has been shown to be
root-specific40. Abundant expression in roots has also been shown
in the ATHK1 gene and some ATRR genes31,39. The similar expression
pattern suggests that some of these components function together
in a certain phosphorelay signaling system. Similarly, some
Arabidopsis response-regulator genes are induced by exogenous
cytokinins, as mentioned above35,36. Whether the expression of the
ATHP genes responds to cytokinin treatment is therefore of interest.
Upstream and downstream of the phosphorelay network:
where is my partner?
Cyanobacterial two-component systems
An increase in the number of the two-component molecules has
raised a question as to which molecules constitute a His-to-Asp
phosphorelay network. To address this issue, several studies have
been conducted as an initial characterization using two strategies,
yeast two-hybrid interaction and in vitro phosphotransfer analysis. Such results provide some preliminary insight into the presumed phosphorelay network in plants, as follows, although they
remain miscellaneous.
Firstly, ETR1 and ERS1 have been demonstrated to interact
directly with CTR1 by both yeast two-hybrid analysis and in vitro
interaction assay43. The ctr1 mutant exhibits constitutive ethylene
responses in the absence of ethylene. Epistasis analysis indicates
that CTR1 acts downstream of ETR1 in the ethylene signal transduction pathway. The CTR1 gene has been cloned by T-DNA tagging and been found to encode a protein kinase that resembles a
Raf protein kinase, a member of MAPKKK (Ref. 44). Sequence
similarity between CTR1 and MAPKKKs suggests that the ethylene-signaling pathway is similar to the yeast osmoregulation pathway. This interaction requires the N-terminal domain of CTR1
and the kinase domain of ETR1 or ERS1. The N-terminal domain
of Raf, which corresponds to the region needed for the interaction
with ETR1 and ERS, has been shown to associate with several
signal transducers, such as Ras and the 14-3-3 proteins, and therefore is believed to function as a regulatory domain. This observation,
together with the direct interaction between ETR1 and CTR1, led
Finally, it would be interesting to discuss the cyanobacterial
two-component systems, because many plant genes for the twocomponent systems might have originated from those of cyanobacteria during endosymbiosis. The two genes for the phytochrome
homolog in cyanobacteria that belong to the two-component histidine kinase are one of the best examples. They are the cph1 gene
of Synechocystis PCC6803 (Refs 46,47) and the rcaE gene of
Fremyella diplosiphon48. The Cph1 protein is a sensor histidine
kinase that has sequence similarity with plant phytochromes in
this N terminus. The Cph1 protein has light-responsive histidine
kinase activity and can bind chromophores to undergo a red/far-red
reversible reaction46,47. Cph1 transfers the phosphoryl group from
histidine to aspartate residues in the Pcp1 protein, a response regulator, and constitutes a typical two-component system. The rcaE
gene product of cyanobacteria F. diplosiphon functions as a
sensor in chromatic adaptation48. The RcaE protein has sequence
similarity with plant phytochromes and ethylene receptors, and constitutes a two-component system with the RcaC protein, a response
regulator.
The sequence analysis of the recently determined entire
genomic DNA database of Synechocystis PCC 6803 has identified
at least 80 open-reading-frames (ORFs) that show a significant
sequence similarity to known members of two-component systems
in bacteria49. Among 26 ORFs identified as putative histidine
kinases, some ORFs have unique structural designs: slr1414,
February 2000, Vol. 5, No. 2
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Table 1. Plant two-component regulators
Two component molecules
Histidine kinases
ETR1
ETR2
EIN4
ERS1
ERS2
CKI1
ATHK1
NR
LeETR1 (= eTAE1)
LeETR2 (= TFE27)
Cm-ETR1
Cm-ERS1
RP-ERS1
DC-ERS1
DC-ERS2
Plants
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Tomato
Tomato
Tomato
Cucumis melo
Cucumis melo
Rumex palustris
Dianthus caryophyllus
Dianthus caryophyllus
Input signals
Structurea
Ref.
Ethylene
TM–KD–RD
2
Ethylene
TM–KD(H/E)–RD 15
Ethylene
TM–KD–RD
14
Ethylene
TM–KD
19
Ethylene
TM–KD(H/D)
14
Cytokinin
TM–KD– –RD
30
Osmorarity?
TM–KD– –RD
31
Ethylene
TM–KD
23
Ethylene?
TM–KD–RD
25,27
Ethylene?
TM–KD–RD
26,27
Ethylene?
TM–KD–RD
28
Ethylene?
TM–KD
28
Ethylene?
TM–KD
29
Ethylene?
TM–KD
50
Ethylene?
TM–KD
51
Phosphorelay
intermediates
ATHP1 (=AHP2)
ATHP2 (= AHP3)
ATHP3 (= AHP1)
ZmHP2
Arabidopsis
Arabidopsis
Arabidopsis
Maize
HPt
HPt
HPt
HPt
40,41
40,41
40,41
42
Response regulators
Type A
ARR3
ARR4 (= ATRR1, IBC7)
ARR5 (= ATRR2, IBC6)
ARR6
ARR7
ARR8 (= ATRR3)
ARR9 (= ATRR4)
ZmRR1
ZmRR2
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Maize
Maize
RD–
RD–
RD–
RD–
RD–
RD–
RD–
RD–
RD–
33
33,35,39
33,35,39
33
33
32,39
32,39
37
42
kinase and receiver domains at both termini,
flanked by a putative input domain. In
many cases of E. coli, cognate kinaseregulator pairs are located next to each
other and work together in specific adaptive responses. Similarly, in cyanobacteria,
some sets of the identified ORFs are organized in the genome in the same manner.
Thus, cyanobacterial two-component systems have several unusual structures that
are supposed to constitute more complicated and sophisticated signaling circuits.
These intriguing findings might provide us
with helpful clues to understand the molecular mechanism underlying the multistep
His-to-Asp phosphorelay signaling in
plants, because cyanobacteria are evolutionarily related to higher plants. In this regard,
it is noteworthy that database searches
have revealed that an ORF, slr1212, from
Synechocystis shows sequence homology
to the ethylene-binding domain of ETR1
but lacks a histidine kinase domain22.
Ethylene-binding assays in vivo indicate
that the gene product exhibits ethylenebinding activity. The presence of both the
functional ethylene-binding domain and
the histidine kinase domain in the cyanobacterial genome raises interesting questions about the evolutionary origin of
ethylene receptors in higher plants.
Conclusion and perspectives
Many two-component regulator genes have
been identified in higher plants (Table 1).
They are histidine kinases, response regulators and phosphorelay intermediates containing an HPt domain. Among them
Type B
hybrid-type histidine kinases for ethylene
ARR1
Arabidopsis
RD– – –
34
receptors have been analyzed extensively
ARR2
Arabidopsis
RD– – –
34
(Fig. 3). In Arabidopsis, ETR1 functions as
ARR10
Arabidopsis
RD– – –
32
an ethylene receptor and is a negative reguARR11
Arabidopsis
RD– – –
32
lator in the signal transduction cascade.
ARR12
Arabidopsis
RD– – –
32
ARR13
Arabidopsis
RD– – –
32
ETR1 has functional homologs, which
ARR14
Arabidopsis
RD– – –
32
indicates the redundant process of signal
perception in signaling. However, the
a
‘Structure’ indicates the domain constructions of the listed proteins: TM, transmembrane region;
downstream cascades remain unclear. Is
KD, kinase domain; RD, receiver domain; HPt, histidine-containing phosphotransfer domain. One dash
any MAP kinase cascade involved in the
corresponds to ~50–100 amino acids in length. (H/D) and (H/E) represent the amino acid substitutions of
ethylene signaling? Are there any phosphohistidine (H) to aspartate (D) and to glutamate (E), respectively.
relay intermediates and response regulators
that function in ethylene signaling? CTR1
encodes a Raf protein kinase homolog and
slr1285 and sll0094 have several amino acid criteria for a typical interacts with ETR1, which suggest the existence of a different
kinase domain but do not have invariant histidine residues. ORF type of phosphorelay system in higher plants. Histidine kinases
slr0073 contains an invariant histidine residue but not other criti- CKI1 and CKI2 also function in cytokinin signaling. Several
cal amino acids, in contrast with slr1414, slr1285 and sll0094. The response regulators can be suggested to function in cytokinin sigORF slr1475 lacks any presumed input domains. Most of the 38 naling based on their cytokinin-inducible gene expression. Their
ORFs identified as putative response regulators have a typical functions as cytokinin receptors have not been determined.
structural design. However, seven ORFs (slr1042, slr1037, sll1292, Recently, an Arabidopsis hybrid-type histidine kinase, ATHK1,
slr2024, slr11982, slr0474 and sll0039) lack any presumed output which functions as an osmosensor in yeast, has been reported, but
domains. Of 16 ORFs identified as putative hybrid histidine its function in plants as an osmosensor remains unclear. The molkinases, several of the ORFs have intriguing structural character- ecular functions of these putative sensors or receptors will be
istics that have never been found in E. coli. For instance, slr0322 characterized precisely in the near future, and members of their
has two tandemly located receiver domains and slr0222 contains two-component systems will be identified based on biochemical
72
February 2000, Vol. 5, No. 2
trends in plant science
Reviews
and genetic analyses. In the next 4 months, the Arabidopsis
genome sequence will be determined, and all the putative genes
for two-component systems will be identified in the database.
Reverse genetics as well as forward genetics are powerful tools
for analyzing the various functions of the two-component regulators
in plants. Classical biochemical analyses are also important for
analyzing the molecular mechanisms of signaling processes.
The two-component system is now thought to play an important
role in sensing environmental stimuli and growth regulators in
higher plants. Other higher plant receptors are the receptor kinases,
which are involved in signaling during pathogen infection. Receptor kinases contain a leucine-rich repeat domain, which is thought
to be involved in ligand binding. By contrast, animals do not have
two-component systems. They have large tyrosine kinases that
function as receptors. These observations strongly suggest that the
molecules involved in signal perception in plants and animals
have different origins. Some plant two-component regulators
appear to have come from cyanobacteria. Cyanobacterial genes
for two-component systems might be transferred to the nuclear
genome of the host cells during endosymbiosis. The plant twocomponent system could have originated from the common
ancestors of yeast and fungi. After completion of Arabidopsis
genome sequencing, comparative genomics of Arabidopsis and
C. elegans should prove the existence of different sets of genes for
signal transduction processes in plants and animals.
Finally, a search of currently available ESTs and the genomic
DNA databases in Arabidopsis revealed many unpublished genes
that have sequence similarity to known two-component molecules: sequences showing homology to histidine kinase domains
(AB011485, AC004557, AC007069); sequences showing homology
to receiver domains (N96794, W43046, AB02429, AB011485,
AC004557, AC007069, AB016872, AC005310, AB025641,
AB019231, AB010073); and sequences showing homology to
HPt domains (T42057, Z17687, T43076, AF069441, AC002560,
AB0230465, AC001645).
Acknowledgements
We thank Prof. Takeshi Mizuno and Dr Takashi Hirayama for
critically reading this manuscript. We also thank Dr Tatsuo Kakimoto
for providing us with his unpublished data.
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Takeshi Urao and Kazuko Yamaguchi-Shinozaki are at the
Biological Resources Division, Japan International Research
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Forestry and Fisheries, 1-2 Ohwashi, Tsukuba, Ibaraki 305, Japan;
Kazuo Shinozaki* is at the Laboratory of Plant Molecular Biology,
Institute of Physical and Chemical Research (RIKEN), Tsukuba
Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
*Author for correspondence (tel 181 298 36 4359;
fax 181 298 36 9060; e-mail [email protected]).
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