Two-Component Systems in Plant Signal Transduction Takeshi Urao, Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki

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.

ll living organisms have diverse and sophisticated signal- A multistep His-to-Asp phosphorelay: more than ing strategies to recognize and respond to their environ- two components Amental conditions. Protein phosphorylation is a key Such a simple His-to-Asp phosphorelay is not the only two-com- mechanism for intracellular signal transduction in both eukaryotic ponent system. In some cases, two-component systems include and prokaryotic cells. This process is catalyzed by protein additional signaling modules or motifs and constitute more kinases, and these are classified into three major groups based on complicated phosphorelay circuits (Fig. 1). This additional signal- their substrate specificities: serine/threonine kinases, tyrosine ing domain, known as the histidine-containing phosphotransfer kinases and histidine kinases. Histidine kinases in bacteria play (HPt) domain, was first uncovered in an E. coli anaerobic sensor key roles in sensing and transducing extracellular signals in- ArcB. For instance, an osmosensory two-component system in cluding chemotactic factors, changes in osmolarity, and nutrient Saccharomyces cerevisiae consists of three signaling molecules, deficiency. This signal transduction system is mediated by SLN1, YPD1 and SSK1 (Ref. 3). The histidine kinase SLN1 acts phosphotransfer between two types of signal transducers and is as a transmembrane osmosensor. SLN1 has both a kinase and a therefore referred to as the ‘two-component system’1. The two- receiver domain within the same molecule. This type of histidine component system has been well defined in bacteria, and, in kinase is referred to as ‘hybrid histidine kinase’. At normal osmo- 1993, the existence of a bacterial-type histidine kinase was re- larity, SLN1 is activated to autophosphorylate a histidine residue ported in yeast and in Arabidopsis2,3. Since then, many genes within the kinase domain using ATP as a phosphodonor. The encoding two-component regulators have been identified in phosphoryl group is transferred sequentially by a phosphorelay plants, yeast and Dictyostelium3,4. However, the existence of two- reaction to an aspartate residue within the receiver domain, and component regulators in animals has not been reported. In this then to a histidine residue in the intermediary molecule YPD1, review, we focus on the two-component regulators and their and finally to an aspartate residue within the receiver domain of function in plants. the response regulator SSK1. The phosphorylated form of SSK1 is incapable of activating an osmosensing HOG1 MAPK cascade. A simple His-to-Asp phosphorelay: two components By contrast, under conditions of high osmolarity, SLN1 is inacti- Typically, the two-component system is composed of a sensory vated, enabling unphosphorylated SSK1 to accumulate, which histidine kinase and a response regulator (Fig. 1). The histidine leads to the activation of the HOG1 MAPK cascade. A similar sig- kinase contains an N-terminal input domain and a C-terminal naling system also operates in osmosensing in fission yeast. The kinase domain with an invariant histidine residue. The response response regulator MCS4 regulates a WAK1-WIS1-STY1 MAPK regulator contains an N-terminal receiver domain with an in- pathway in Schizosaccharomyces pombe5. Such a multistep phos- variant aspartate residue and a C-terminal output domain. For phorelay reaction is believed to have the potential advantages of example, in E. coli, osmotic responses are controlled by an providing multiple regulatory checkpoints for signal cross-talk or EnvZ–OmpR two-component system. The input domain of the negative regulation by certain phosphatases. Multistep phospho- sensory histidine kinase EnvZ somehow detects changes in exter- relays are known to be involved in anaerobic regulation in E. coli, nal osmolarity and modulates intrinsic kinase and phosphatase in sporulation in Bacillus subtilis, and in virulence control in activities. High osmolarity promotes autophosphorylation of a Bordetella pertussis. The existence of multistep phosphorelay histidine residue within its kinase domain, followed by transfer reactions in both prokaryotes and eukaryotes suggests that similar of the phosphoryl group to an aspartate residue within the mechanisms might be more widely used. receiver domain of the cognate response regulator OmpR. By contrast, low osmolarity promotes dephosphorylation of the Link between prokaryotic two-component system phosphorylated OmpR. The phosphorylation state of OmpR and eukaryotic signal transduction system alters the DNA-binding activity of the output domain to control The best documented linkage between prokaryotic and eukaryotic the transcription of target genes. Thus, a physical signal (e.g. two-component signal transduction mechanisms is for the an environmental stimulus) can be converted to a biochemical osmoregulation systems of yeast. As mentioned already, in reaction, termed ‘His-to-Asp phosphorelay’, by two-component S. cerevisiae, exposure to high osmolarity activates the HOG1 signaling systems. MAPK cascade through the SLN1-YPD1-SSK1 two-component

system. At high osmolarity, the unphosphorylated response Histidine Phosphorelay Response regulator SSK1 activates SSK2 and SSK22 (MAPKKK) by direct kinase intermediate regulator protein–protein interaction. The activated SSK2 and SSK22 phos- phorylates PBS2 (MAPKK), and HOG1 (MAPK) is activated. (a) Furthermore, it should be noted that, in addition to SLN1, another P P osmosensor, SHO1, is known in budding yeast3. SHO1 is not a H D member of the two-component family, but it acts as a transmem- EnvZ OmpR brane osmosensor, activating PBS2 MAPKK by direct interaction between the SH3 domain of SHO1 and the proline-rich region (b) of PBS2. SLN1 and SHO1 appear to have a different salt concen- P P P P tration dependency and a different time course for providing an optimal response to osmolarity changes. In yeast, multiple MAPK H D H D cascades are involved in the responses to a variety of external YPD1 SSK1 SLN1 stimuli. For example, STE11 (MAPKKK) shares two independent Trends in Plant Science pathways, an osmosensing SHO1–HOG1 pathway and a pheromone- Fig. 1. Two types of phosphorelay signaling in two-component response FUS3/KSS1 pathway. Nevertheless,osmotic stress, which systems. The input domain is depicted in pink, the transmembrane leads to the activation of the HOG1 MAPK cascade, never acti- region in grey, the kinase domain in blue, the receiver domain in vates the FUS3/KSS1 MAPK cascade. Thus, the two pathways light green, the histidine-containing phosphotransfer domain in respond to a specific signal, and function independently. This dark green and the output domain in purple. H represents histidine functional separation is achieved by a scaffold protein that prevents and D represents aspartate. (a) Single step His-to-Asp phospho- undesirable signals entering and interfering with the pathway. In relay. The sensory kinase EnvZ recognizes a high osmolarity con- dition and autophosphorelates a histidine residue in the kinase any event, it should be emphasized that the prokaryotic-like two- domain. The phosphoryl group is then transferred to an aspartate component signaling mechanism can be linked downstream to typi- residue in the receiver domain of the response regulator OmpR. cal eukaryotic (MAPK) signaling cascades, in yeast. This intriguing The phosphorylated OmpR promotes its DNA-binding activity view is further supported by recent findings that two-component and activates the transcription of high osmolarity-responsive systems in Dictyostelium play important roles in signal recognition genes. (b) Multistep His-to-Asp phosphorelay. Under conditions and development, as described below. of high osmolarity, the sensory kinase SLN1 autophosphorylates A gene, dokA, encoding a hybrid histidine kinase has been and blocks the activation of the osmoregulatory HOG1 MAPK cloned from Dictyostelium6. The dokA deletion mutants show a cascade through a multistep phosphorelay between an aspartate dramatic reduction in viability after exposure to medium of high residue in the receiver domain, a histidine residue in the phos- osmolarity and inhibition of growth and development under less phorelay intermediate YPD1, and an aspartate residue in the receiver domain of the response regulator SSK1. By contrast, high stringent osmolarity conditions. DokA, unlike EnvZ and SLN1, osmolarity inactivates SLN1, resulting in the accumulation of does not contain extensive hydrophobic regions, suggesting a unphosphorylated SSK1. soluble intracellular signal transducer. The signals sensed by DokA might be either concentrated intracellular solutes or sec- ondary molecules, such as polyamines and trehalose derivatives. Alternatively, DokA might perceive osmolarity changes by indirectly monitoring the medium using a separate sensory protein. CKI1 The development of the Dictyostelium fruiting body is controlled ATHK1 7 ETR1 by an RdeA–RegA two-component system . RegA has two ETR2 ERS1 functional domains, one is homologous to a mammalian cAMP EIN4 ERS2 phosphodiesterase and the other to bacterial response regulators; subsequent analysis has revealed that phosphorylation of the ARR3 ARR1 RegA receiver domain stimulates the output activity of the phos- H H H ATHP1 (AHP2) ARR4 ARR2 phodiesterase domain8. RdeA has weak homology to the yeast ATHP2 (AHP3) (ATRR1, IBC7) ARR10 ARR5 phosphorelay intermediate YPD1, which might complement an ATHP3 (AHP1) ARR11 9 D D (ATRR2, IBC6) ARR12 rdeA null mutant . Indeed, direct phosphotransfer from RdeA to ARR6 H ARR13 RegA has been demonstrated in vitro, and it therefore appears that ARR7 ARR14 ARR8 (ATRR3) RdeA is an immediate upstream factor for RegA. Although a Histidine ARR9 (ATRR4) histidine kinase that functions together in this signaling pathway kinases Phosphorelay has not been identified, a multistep phosphorelay two-component intermediate D D system might play a role in controlling the development of the fruiting body in Dictyostelium. In this case, it is interesting to Response emphasize that the RdeA–RegA two-component system appears regulators 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 Histidine kinases as ethylene receptors in Arabidopsis domain is depicted in pink, the transmembrane region in grey, the Several histidine kinases have been cloned and shown to be kinase domain in blue, the receiver domain in light green, the involved in the perception of plant hormones and environmental histidine-containing phosphotransfer domain in dark green and the signals. In Arabidopsis, several ethylene response mutants have output domain in purple. H represents histidine and D represents been isolated and extensively characterized. The etr1, etr2 and aspartate. ein4 mutants have dominant ethylene insensitivity and are supposed to act at an early step in ethylene signal transduction4,10–13.

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The ETR1 gene was first isolated by map- 2 based cloning . Subsequently, ETR2 and Cu RAN1 EIN4 were also cloned14,15. Sequence analy- ETR1 sis has revealed that three genes en- ERS1 code hybrid histidine kinases, with three Air ETR2 CTR1 EIN2 EIN3 OFF 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 16–18 Ethylene ETR2 CTR1 EIN2 EIN3 ON within the transmembrane region . ERS2 These results, together with the etr1 mutant EIN4 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 ETR1-related genes, ERS1 and ERS2, have in an active state in the absence of ethylene and activates CTR1 directly or indirectly. been cloned from Arabidopsis based on the The activated CTR1 represses the ethylene responses by inactivation of the downstream sequence similarity with ETR1, and shown components EIN2 and EIN3. Binding ethylene to ETR1 modulates the activity to an inactive to be involved in ethylene perception14,19. form. The inactivated ETR1 inactivates CTR1 and consequently permits ethylene responses. Both proteins contain a kinase domain but Thus, ETR1 and CTR1 function as negative regulators in ethylene action. RAN1 participates in delivering copper ions to functional ethylene receptors. In the etr1 mutant, the gain- not 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 in- the 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 up- of-function) could not be obtained by the previous genetic regulation of ethylene receptors is likely to be involved in a mech- screens. 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 pheno- receptors 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 copper- To address this issue, loss-of-function mutants (knock-out) of four transporting activity. Based on in planta demonstration using trans- members, 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

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germination. More recently, homologs of ETR1 and ERS1, Cm- and accumulated under the conditions of high-salinity and low ETR1 and Cm-ERS1, respectively, have been isolated from temperature. The high level of expression in roots suggests that melon28. The level of Cm-ERS1 mRNA increases during early ATHK1 is necessary for efficient sensing of environmental sig- fruit development and decreases at the end of fruit enlargement, nals, such as high-salinity and drought. What are the advantages whereas the level of Cm-ETR1 mRNA is high in the seeds and of the up-regulation of osmosensors? If increased osmotic pres- placenta of developing and in fully enlarged fruit. During fruit sure causes a conformational change in ATHK1 and consequently ripening, the level of Cm-ERS1 mRNA increases slightly, its kinase activity changes to an inactive state, the newly produced whereas the level of the Cm-ETR1 mRNA markedly increases in ATHK1 under conditions of high-osmolarity would also be inac- the pericarp. Thus, the gene expression of the two putative ethyl- tive in the membrane, leading to the activation of downstream ene receptors is differentially regulated with the developmental pathways. Accumulation of inactive ATHK1 appears to be re- stage and in the tissue of mature fruit. Genomic Southern blot quired to enhance the responses or to reduce the responses quickly analysis indicated the presence of an additional related gene in by the reactivation of ATHK1 kinase activity when osmotic stress melon. The flooding response is also known to be initiated by eth- is relieved. ylene. As the first step in understanding the molecular mechanism of the flooding responses, RP-ERS1, a homolog of Arabidopsis Response regulators in higher plants ERS1 and tomato NR, has been cloned from Rumex palustris, a In addition to the histidine kinases mentioned already, many re- flooding-tolerant plant29. RP-ERS1 gene expression is induced by sponse regulators have also been isolated in Arabidopsis. Recently, flooding, ethylene, carbon dioxide and low oxygen. Some addi- it has been proposed that they could be classified into two groups, tional homologous genes appear to exist in R. palustris. Thus, type-A and type-B, based on their architecture32. The type-A re- plants, at least climacteric fruits, appear to have two types of sponse regulators are mainly composed of a receiver domain and ethylene receptor, an ethylene-inducible type (ERS1-like family) short N- and C-terminal extensions, whereas the type-B response and a constitutive-expression type (ETR1-like family), which regulators have a receiver domain and a largely extended C-terminal might lead to distinct ethylene sensitivities. region that is supposed to be an output domain (Fig. 2). ARR3-7, members of the type-A group, were initially cloned in Arabidopsis Histidine kinases involved in cytokinin signaling using an EST database33. By using in vitro phosphotransfer analy- Plants appear to use a histidine kinase as a cytokinin receptor. In sis, ARR3 has been shown to accept a phosphoryl group from a Arabidopsis, several mutants (cki1-1, -2, -3, and -4, and cki2) that phospho-HPt domain of an E. coli hybrid, histidine kinase ArcB. show typical cytokinin responses in the absence of exogenous In addition, overexpression of ARR3 results in the reduction of cytokinin have been isolated by activation tagging30. A gene, the OmpC gene expression, suggesting that ARR3 competes with CKI1, has been cloned and shown to encode a novel hybrid histi- OmpR, a transcriptional activator for the OmpC gene, by titrating dine kinase (Fig. 2). Transformation of Arabidopsis wild-type the phosphoryl group from the HPt domain of ArcB. It is thus dem- plants with CKI1 under the control of a single CaMV 35S pro- onstrated that ARR3 has a functional receiver domain as a phospho- moter has confirmed that cytokinin-independent growth is caused acceptor. ARR1 and ARR2, members of the type-B group, have by the overproduction of CKI1. Ectopic expression of CKI1 could been cloned subsequently and shown to contain a large extended lead to the overestimation of low levels of endogenous cytokinin, C-terminal region, a part of which has a weak homology to single which are usually unable to trigger specific responses. A gene for Myb DNA-binding proteins from potato34. Indeed, their C-terminal CKI2 has been isolated and shown to encode a hybrid histidine regions are rich in glutamate and proline, which is one of the char- kinase (T. Kakimoto, pers. commun.). The structure of CKI2 is acteristics of eukaryotic transcriptional activation domains. Simi- different from that of CKI1. CKI1 and CKI2 might act as cyto- larity to the Myb DNA-binding proteins has also been found in kinin receptors with distinct signaling mechanisms. other members of the type-B group32. Although the DNA-binding activity of the type-B response regulators has not been demon- Hybrid histidine kinase functions as an osmosensor strated, the conservation of this region might imply the functional in plants importance of this region in the regulation of biological function, Recently, the possible involvement of a histidine kinase in such as gene expression. osmosensing in plants has been shown. A hybrid histidine kinase The two classes of response regulators differ not only in their ATHK1 has been cloned from Arabidopsis31. ATHK1 contains structural features, but also in their expression patterns and bio- two hydrophobic transmembrane regions adjacent to a putative chemical activities. The genes for the type-A response regulators, extracellular domain in the N-terminal half, suggesting functional ARR3, ARR4 (ATRR1, IBC7), ARR5 (ATRR2, IBC6), ARR6 and similarity with the yeast osmosensor SLN1 (Fig. 2). This possibil- ARR7, are induced by exogenous cytokinins, but not by any other ity was demonstrated by analyzing both the sensing (input) and the plant hormones35,36. Moreover, re-application of nitrate to N- catalytic (output) activities of ATHK1 using yeast osmosensing- starved plants also results in the accumulation of their transcripts, defective mutants. ATHK1 can suppress the sln1-ts (sln1-4) as previously observed in maize ZmRR1 (Ref. 37). These results mutant. By contrast, the substitution of either putative phosphory- suggest that these cytokinin-responsive ARRs are involved, at lation site, His or Asp, failed to complement the sln1-ts mutant, least in part, in the nitrate signal transduction mediated by indicating that ATHK1 acts as a histidine kinase in yeast and that cytokinin in Arabidopsis. In this regard, it is noteworthy that an ATHK1 is in an active state in the absence of external signals (e.g. Arabidopsis histidine kinase CKI1 is implicated to function as a high osmolarity). Moreover, ATHK1 allowed a yeast mutant lack- cytokinin receptor. Some of these ARRs might work together with ing both osmosensors, SLN1 and SHO1, to activate HOG1 and to CKI1 in a certain cytokinin-sensing two-component system. In grow normally under conditions of high osmolarity, suggesting contrast with the type-A ARRs, ARR1, ARR2 and ARR10, mem- that the ATHK1 activity changed to an inactive state from an bers of the type-B group, do not respond to these treatments38. active state in response to the increase in external osmolarity. Thus, two types of ARRs show distinct cytokinin- and nitrate- Thus, ATHK1 appears to have an ability to sense and transduce a responsiveness. Moreover, the type-A members can be further signal of external osmolarity to the downstream targets in yeast. classified into two subgroups, based on their responsiveness to The ATHK1 mRNA was more abundant in roots than other tissues stress treatments. Among the type-A members, the ATRR1 (ARR4,

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IBC7) and ATRR2 (ARR5, IBC6) genes are induced by high salin- to the hypothesis that CTR1 is a part of an ethylene receptor com- ity and low temperature, whereas the transcript levels of ATRR3 plex and that the activity of CTR1 is regulated by direct interac- and ATRR4 are not affected by any of the stress treatments that tion with the two-component histidine kinases. However, other have been tested39. In in vitro phosphotransfer analysis, ARR3 and two-component molecules, such as response regulators and phos- ARR4, members of the type-A group, have been shown to have an phorelay intermediates, have also been isolated from Arabidopsis. ability to accept a phosphoryl group from the phosphorylated HPt Based on the current two-component system scheme, it is reason- domain of AHP1 and AHP2, Arabidopsis phosphorelay inter- able to suppose that other components are also involved in mediates32. By contrast, ARR10, a member of the type-B group, is the formation of the receptor-CTR1 complex, and that CTR1 incapable of exhibiting phospho-acceptor activity toward any of activity is regulated by output activity initiated by ethylene the AHPs tested. receptor-mediated phosphorelays. Secondly, in vitro phosphotransfer analysis has indicated that Phosphorelay intermediates containing a His-containing ARR3 and ARR4 have an ability to accept a phosphoryl group phosphotransfer domain in higher plants from the phosphorylated HPt domain of AHP1 and AHP2, In Arabidopsis, a third component has simultaneously been whereas ARR10 is incapable of exhibiting a phosphoacceptor cloned by two independent groups40,41. Three Arabidopsis phos- activity toward any AHPs tested32. AHP1 or AHP2 might be an phorelay intermediates, ATHP1 (AHP2), ATHP2 (AHP3) and upstream phosphorelay mediator of ARR3 or ARR4, and they ATHP3 (AHP1), contain an HPt domain with a conserved histi- might function together in a particular two-component system. dine and some invariant residues that are crucial for phosphorelay Two-hybrid screening has identified two closely related proteins, (Fig. 2). These are somewhat similar to the yeast YPD1 in the ATDBP1 and ATDBP2, as proteins that interact with the response sense that they are small polypeptides containing only an HPt regulator ARR4 (Ref. 45). ATDBP1 has sequence similarity with domain. Indeed, functional analysis using a yeast ypd1 mutant has Remorin from potato, which is a membrane-associated and demonstrated that all ATHPs act as phosphorelay intermediates uranide-binding phosphoprotein. ATDBP1 and ATDBP2 might between SLN1 and SSK1 in yeast40. Moreover, the recombinant be a downstream target or an upstream regulator for ARR4, be- AHP1 protein could be phosphorylated by an uncertain hybrid- cause such small response regulators lacking a presumed output type histidine kinase in the E. coli membrane fractions as a phos- domain probably function as an on–off switch molecule at an pho-donor41. Subsequent analysis has revealed that the phosphoryl intermediate step in His-to-Asp phosphorelay. Alternatively, group on the histidine residue of AHP1 is transferred transiently ATDBP1 and ATDBP2 might assist ARR4 in the localization to the to the receiver domain of ARR3 and ARR4 in vitro41. Thus, it has plasma membrane where a certain two-component system works. been shown that AHP1 (ATHP3) exhibits an ability to accept and A phosphorelay network can be assumed by the similarity of transfer the phosphoryl group directly to the response regulators their expression profiles. The ATHP3 gene has been shown to be in Arabidopsis as well as in yeast. Recently, a phosphorelay inter- root-specific40. Abundant expression in roots has also been shown mediate, ZmHP2, has been cloned from maize42. In vitro experi- in the ATHK1 gene and some ATRR genes31,39. The similar expression ments have shown that ZmHP2 is phosphorylated by E. coli inner pattern suggests that some of these components function together membrane vesicles, and that the phosphoryl group on ZmHP2 is in a certain phosphorelay signaling system. Similarly, some transiently transferred to ZmRR1 and ZmRR2 response regulators Arabidopsis response-regulator genes are induced by exogenous from maize. 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 Finally, it would be interesting to discuss the cyanobacterial raised a question as to which molecules constitute a His-to-Asp two-component systems, because many plant genes for the two- phosphorelay network. To address this issue, several studies have component systems might have originated from those of cyano- been conducted as an initial characterization using two strategies, bacteria during endosymbiosis. The two genes for the phytochrome yeast two-hybrid interaction and in vitro phosphotransfer analy- homolog in cyanobacteria that belong to the two-component histi- sis. Such results provide some preliminary insight into the pre- dine kinase are one of the best examples. They are the cph1 gene sumed phosphorelay network in plants, as follows, although they of Synechocystis PCC6803 (Refs 46,47) and the rcaE gene of remain miscellaneous. Fremyella diplosiphon48. The Cph1 protein is a sensor histidine Firstly, ETR1 and ERS1 have been demonstrated to interact kinase that has sequence similarity with plant phytochromes in directly with CTR1 by both yeast two-hybrid analysis and in vitro this N terminus. The Cph1 protein has light-responsive histidine interaction assay43. The ctr1 mutant exhibits constitutive ethylene kinase activity and can bind chromophores to undergo a red/far-red responses in the absence of ethylene. Epistasis analysis indicates reversible reaction46,47. Cph1 transfers the phosphoryl group from that CTR1 acts downstream of ETR1 in the ethylene signal trans- histidine to aspartate residues in the Pcp1 protein, a response regu- duction pathway. The CTR1 gene has been cloned by T-DNA tag- lator, and constitutes a typical two-component system. The rcaE ging and been found to encode a protein kinase that resembles a gene product of cyanobacteria F. diplosiphon functions as a Raf protein kinase, a member of MAPKKK (Ref. 44). Sequence sensor in chromatic adaptation48. The RcaE protein has sequence similarity between CTR1 and MAPKKKs suggests that the ethyl- similarity with plant phytochromes and ethylene receptors, and con- ene-signaling pathway is similar to the yeast osmoregulation path- stitutes a two-component system with the RcaC protein, a response way. This interaction requires the N-terminal domain of CTR1 regulator. and the kinase domain of ETR1 or ERS1. The N-terminal domain The sequence analysis of the recently determined entire of Raf, which corresponds to the region needed for the interaction genomic DNA database of Synechocystis PCC 6803 has identified with ETR1 and ERS, has been shown to associate with several at least 80 open-reading-frames (ORFs) that show a significant signal transducers, such as Ras and the 14-3-3 proteins, and there- sequence similarity to known members of two-component systems fore is believed to function as a regulatory domain. This observation, in bacteria49. Among 26 ORFs identified as putative histidine together with the direct interaction between ETR1 and CTR1, led kinases, some ORFs have unique structural designs: slr1414,

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kinase and receiver domains at both termini, Table 1. Plant two-component regulators flanked by a putative input domain. In many cases of E. coli, cognate kinase- Two component molecules Plants Input signals Structurea Ref. regulator pairs are located next to each other and work together in specific adap- Histidine kinases tive responses. Similarly, in cyanobacteria, ETR1 Arabidopsis Ethylene TM–KD–RD 2 some sets of the identified ORFs are orga- ETR2 Arabidopsis Ethylene TM–KD(H/E)–RD 15 nized in the genome in the same manner. EIN4 Arabidopsis Ethylene TM–KD–RD 14 ERS1 Arabidopsis Ethylene TM–KD 19 Thus, cyanobacterial two-component sys- ERS2 Arabidopsis Ethylene TM–KD(H/D) 14 tems have several unusual structures that CKI1 Arabidopsis Cytokinin TM–KD– –RD 30 are supposed to constitute more compli- ATHK1 Arabidopsis Osmorarity? TM–KD– –RD 31 cated and sophisticated signaling circuits. NR Tomato Ethylene TM–KD 23 These intriguing findings might provide us LeETR1 (= eTAE1) Tomato Ethylene? TM–KD–RD 25,27 with helpful clues to understand the molec- LeETR2 (= TFE27) Tomato Ethylene? TM–KD–RD 26,27 ular mechanism underlying the multistep Cm-ETR1 Cucumis melo Ethylene? TM–KD–RD 28 His-to-Asp phosphorelay signaling in Cm-ERS1 Cucumis melo Ethylene? TM–KD 28 plants, because cyanobacteria are evolution- RP-ERS1 Rumex palustris Ethylene? TM–KD 29 arily related to higher plants. In this regard, DC-ERS1 Dianthus caryophyllus Ethylene? TM–KD 50 DC-ERS2 Dianthus caryophyllus Ethylene? TM–KD 51 it is noteworthy that database searches have revealed that an ORF, slr1212, from Phosphorelay Synechocystis shows sequence homology intermediates to the ethylene-binding domain of ETR1 ATHP1 (=AHP2) Arabidopsis HPt 40,41 but lacks a histidine kinase domain22. ATHP2 (= AHP3) Arabidopsis HPt 40,41 Ethylene-binding assays in vivo indicate ATHP3 (= AHP1) Arabidopsis HPt 40,41 that the gene product exhibits ethylene- ZmHP2 Maize HPt 42 binding activity. The presence of both the functional ethylene-binding domain and Response regulators the histidine kinase domain in the cyano- Type A ARR3 Arabidopsis RD– 33 bacterial genome raises interesting ques- ARR4 (= ATRR1, IBC7) Arabidopsis RD– 33,35,39 tions about the evolutionary origin of ARR5 (= ATRR2, IBC6) Arabidopsis RD– 33,35,39 ethylene receptors in higher plants. ARR6 Arabidopsis RD– 33 ARR7 Arabidopsis RD– 33 Conclusion and perspectives ARR8 (= ATRR3) Arabidopsis RD– 32,39 Many two-component regulator genes have ARR9 (= ATRR4) Arabidopsis RD– 32,39 been identified in higher plants (Table 1). ZmRR1 Maize RD– 37 They are histidine kinases, response regu- ZmRR2 Maize RD– 42 lators and phosphorelay intermediates containing an HPt domain. Among them Type B ARR1 Arabidopsis RD– – – 34 hybrid-type histidine kinases for ethylene ARR2 Arabidopsis RD– – – 34 receptors have been analyzed extensively ARR10 Arabidopsis RD– – – 32 (Fig. 3). In Arabidopsis, ETR1 functions as ARR11 Arabidopsis RD– – – 32 an ethylene receptor and is a negative regu- ARR12 Arabidopsis RD– – – 32 lator in the signal transduction cascade. 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 histidine (H) to aspartate (D) and to glutamate (E), respectively. ethylene signaling? Are there any phosphorelay 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 sig- ORF 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 mol- kinases, 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

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and genetic analyses. In the next 4 months, the Arabidopsis 10 Chang, C. (1996) The ethylene signal transduction pathway in Arabidopsis: an genome sequence will be determined, and all the putative genes emerging paradigm? Trends Biochem. Sci. 21, 129–133 for two-component systems will be identified in the database. 11 Bleecker, A.B. and Schaller, G.E. (1996) The mechanism of ethylene Reverse genetics as well as forward genetics are powerful tools perception. Plant Physiol. 111, 653–660 for analyzing the various functions of the two-component regulators 12 Kieber, J.J. (1997) The ethylene response pathway in Arabidopsis. Annu. in plants. Classical biochemical analyses are also important for Rev. Plant Physiol. Plant Mol. Biol. 48, 277–296 analyzing the molecular mechanisms of signaling processes. 13 Bleecker, A.B. (1999) Ethylene perception and signaling: an evolutionary The two-component system is now thought to play an important perspective, Trends Plant Sci. 269–274 role in sensing environmental stimuli and growth regulators in 14 Hua, J. et al. (1998) EIN4 and ERS2 are members of the putative ethylene higher plants. Other higher plant receptors are the receptor kinases, receptor gene family in Arabidopsis. Plant Cell 10, 1321–1332 which are involved in signaling during pathogen infection. Recep- 15 Sakai, H. et al. (1998) ETR2 is an ETR1-like gene involved in ethylene tor kinases contain a leucine-rich repeat domain, which is thought signaling in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 95, 5812–5817 to be involved in ligand binding. By contrast, animals do not have 16 Schaller, G.E. et al. (1995) The ethylene response mediator ETR1 two-component systems. They have large tyrosine kinases that from Arabidopsis forms a disulfide-linked dimer. J. Bio. Chem. 270, function as receptors. These observations strongly suggest that the 12526–12530 molecules involved in signal perception in plants and animals 17 Schaller, G.E. and Bleecker, A.B. (1995) Ethylene-binding sites generated in have different origins. Some plant two-component regulators yeast expressing the Arabidopsis ETR1 gene. Science 270, 1809–1811 appear to have come from cyanobacteria. Cyanobacterial genes 18 Gamble, R.L. et al. (1998) Histidine kinase activity of the ETR1 ethylene for two-component systems might be transferred to the nuclear receptor from Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 95, 7825–7829 genome of the host cells during endosymbiosis. The plant two- 19 Hua, J. et al. (1995) Ethylene insensitivity conferred by Arabidopsis ERS component system could have originated from the common gene. Science 269, 1712–1714 ancestors of yeast and fungi. After completion of Arabidopsis 20 Hua, J. and Meyerowitz, E.M. (1998) Ethylene responses are negatively genome sequencing, comparative genomics of Arabidopsis and regulated by a receptor gene family in Arabidopsis thaliana. Cell 94, 261–271 C. elegans should prove the existence of different sets of genes for 21 Hirayama, T. et al. (1999) RESPONSIVE-TO-ANTAGONIST 1, a signal transduction processes in plants and animals. Menkens/Wilson disease-related copper transporter, is required for ethylene Finally, a search of currently available ESTs and the genomic signaling in Arabidopsis. Cell 97, 383–393 DNA databases in Arabidopsis revealed many unpublished genes 22 Rodríguez, F.I. et al. (1999) A copper cofactor for ethylene receptor ETR1 that have sequence similarity to known two-component mol- from Arabidopsis. Science 283, 996–998 ecules: sequences showing homology to histidine kinase domains 23 Wilkinson, J.Q. et al. (1995) An ethylene-inducible component of signal (AB011485, AC004557, AC007069); sequences showing homology transduction encoded by Never-ripe. Science 270, 1807–1809 to receiver domains (N96794, W43046, AB02429, AB011485, 24 Yen, H-C. et al. (1995) The tomato Never-ripe locus regulates ethylene- AC004557, AC007069, AB016872, AC005310, AB025641, inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 AB019231, AB010073); and sequences showing homology to gene. Plant Physiol. 107, 1343–1353 HPt domains (T42057, Z17687, T43076, AF069441, AC002560, 25 Zhou, D. et al. (1996) The mRNA for an ETR1 homologue in tomato is AB0230465, AC001645). constitutively expressed in vegetative and reproductive tissues. Plant Mol. Biol. 30, 1331–1338 Acknowledgements 26 Zhou, D. et al. (1996) Molecular cloning of a tomato cDNA (Accession no. We thank Prof. Takeshi Mizuno and Dr Takashi Hirayama for U4276) encoding an ethylene receptor. Plant Physiol. 110, 1435 critically reading this manuscript. We also thank Dr Tatsuo Kakimoto 27 Lashbrook, C.C. et al. (1998) Differential regulation of the tomato ETR gene for providing us with his unpublished data. family throughout plant development. Plant J. 15, 243–252 28 Sato-Nara, K. et al. (1999) Stage- and tissue-specific expression of ethylene References receptor homolog genes during fruit development in muskmelon. Plant 1 Parkinson, J.S. and Kofoid, E.C. (1992) Communication modules in bacterial Physiol. 119, 321–329 signaling proteins. Annu. Rev. Genet. 26, 71–112 29 Vriezen, W.H. et al. (1997) A homolog of the Arabidopsis thaliana ERS 2 Chang, C. et al. (1993) Arabidopsis ethylene-response gene ETR1: similarity gene is actively regulated in Rumex palustris upon flooding. Plant J. 11, of product to two-component regulators. Science 262, 539–544 1265–1271 3 Wurgler-Murphy, S.M. and Saito, H. (1997) Two-component signal 30 Kakimoto, T. (1996) CKI1, a histidine kinase homolog implicated in cytokinin transducers and MAPK cascades. Trends Biochem. Sci. 22, 172–176 signal transduction. Science 274, 982–985 4 Chang, C. and Stewart, R. (1998) The two-component system. Plant Physiol. 31 Urao, T. et al. (1999) A transmembrane hybrid-type histidine kinase in 117, 723–731 Arabidopsis functions as an osmosensor. Plant Cell 11, 1743–1754 5 Shieh, J-C. et al. (1997) The Mcs4 response regulator coordinately controls 32 Imamura, A. et al. (1999) Compilation and characterization of Arabidopsis the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast response regulators implicated in His–Asp phosphorelay signal transduction. cell cycle. Genes Dev. 11, 1008–1022 Plant Cell Physiol. 40, 733–742 6 Schuster, S.C. et al. (1996) The hybrid histidine kinase DokA is part of 33 Imamura, A. et al. (1998) Response regulators implicated in His-to-Asp the osmotic response system of Dictyostelium. EMBO J. 15, phosphotransfer signaling in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 95, 3880–3889 2691–2696 7 Thomason, P. et al. (1999) Taking the plunge: terminal differentiation in 34 Sakai, H. et al. (1998) Two-component response regulators from Arabidopsis Dictyostelium. Trends Genet. 15, 15–19 thaliana contain a putative DNA-binding motif. Plant Cell Physiol. 39, 8 Thomason, P.A. et al. (1998) An intersection of the cAMP/PKA and two- 1232–1239 component signal transduction systems in Dictyostelium. EMBO J. 17, 35 Brandstatter, I. and Kieber, J.J. (1998) Two genes with similarity to bacterial 2838–2845 response regulators are rapidly and specifically induced by cytokinin in 9 Chang, W.T. et al. (1998) Evidence that the RdeA protein is a component of a Arabidopsis. Plant Cell 10, 1009–1019 multistep phosphorelay modulating rate of development in Dictyostelium. 36 Taniguchi, M. et al. (1998) Expression Arabidopsis response regulator EMBO J. 17, 2809–2816 homologs is induced by cytokinins and nitrate. FEBS Lett. 429, 259–262

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37 Sakakibara, H. et al. (1998) A response-regulator homologue possibly 46 Hughes, J. et al. (1997) A prokaryotic phytochrome. Nature 386, 663 involved in nitrogen signal transduction mediated by cytokinin in maize. Plant 47 Yeh, K-C. et al. (1997) A cyanobacterial phytochrome two-component J. 14, 337–344 light sensory system. Science 277, 1505–1508 38 Kiba, T. et al. (1999) Differential expression of genes for response regulators in 48 Kehoe, D.M. and Grossman, A.R. (1996) Similarity of a chromatic response to cytokinins and nitrate in Arabidopsis. Plant Cell Physiol. 40, 767–771 adaptation sensor to phytochrome and ethylene receptors. Science 273, 39 Urao, T. et al. (1998) Stress-responsive expression of genes for two- 1409–1412 component response regulator-like proteins in Arabidopsis thaliana. FEBS 49 Mizuno, T. et al. (1996) Compilation of all genes encoding bacterial Lett. 427, 175–178 two-component signal transducers in the genome of the cyanobacterium, 40 Miyata, S. et al. (1998) Characterization of genes for two-component Synechocystis sp. strain PCC 6803. DNA Res. 3, 401–414 phosphorelay mediators with a single HPt domain in Arabidopsis thaliana. 50 Chang, Y-E. et al. (1997) cDNA sequence of a putative ethylene receptor FEBS Lett. 437, 11–14 from carnation petals. Plant Physiol. Plant gene register (PGR97-144) 41 Suzuki, T. et al. (1998) Histidine-containing phosphotransfer (HPt) signal 51 Shibuya, K. et al. (1998) A cDNA encoding a putative ethylene receptor transducers implicated in His-to-Asp phosphorelay in Arabidopsis. Plant Cell related to petal senescence in carnation. Plant Physiol. Plant gene register Physiol. 39, 1258–1268 (PGR98-019) 42 Sakakibara, H. et al. (1999) His–Asp phosphotransfer possibly involved in the nitrogen signal transduction mediated by cytokinin in maize: molecular cloning of cDNAs for two-component regulatory factors and demonstration of phosphotransfer activity in vitro. Plant Mol. Biol. 41, 563–573 Takeshi Urao and Kazuko Yamaguchi-Shinozaki are at the 43 Clark, K.L. et al. (1998) Association of the Arabidopsis CTR1 Raf-like kinase Biological Resources Division, Japan International Research with the ETR1 and ERS ethylene receptors. Proc. Natl. Acad. Sci. U. S. A. 95, Center for Agricultural Science (JIRCAS), Ministry of Agriculture, 5401–5406 Forestry and Fisheries, 1-2 Ohwashi, Tsukuba, Ibaraki 305, Japan; 44 Kieber, J.J. et al. (1993) CTR1, a negative regulator of the ethylene response Kazuo Shinozaki* is at the Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Tsukuba pathway in Arabidopsis, encodes a member of the raf family of protein Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. kinases. Cell 72, 427–441 45 Yamada, H. et al. (1998) An Arabidopsis protein that interacts with the *Author for correspondence (tel ϩ81 298 36 4359; cytokinin-inducible response regulator, ARR4, implicated in the His–Asp fax ϩ81 298 36 9060; e-mail [email protected]). phosphorelay signal transduction. FEBS Lett. 436, 76–80

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