- Email: [email protected]
Ethylene perception and signaling: an evolutionary perspective
trends in plant science reviews 43 Masson, J.E. and Paszkowski, J. (1997) Arabidopsis thaliana mutants altered in homologous recombination, Proc. Natl. Acad. Sci. U. S. A. 94, 11731–11735 44 Revenkova, E. et al. (1999) Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress, EMBO J. 18, 490–499 45 Smith, K.N. et al. (1996) Arabidopsis thaliana RAD51 homolog (AtRAD51), complete coding sequence. EMBL Accession No. U43528 46 Doutriaux, M.P. et al. (1998) Isolation and characterization of the RAD51 and DMC1 homologs from Arabidopsis thaliana, Mol. Gen. Genet. 257, 283–291 47 Stassen, N.Y. et al. (1997) Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs, Curr. Genet. 31, 144–157 48 Klimyuk, V.I. and Jones, J. (1997) AtDMC1, the Arabidopsis homologue of yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression, Plant J. 11, 1–14
49 Kobayashi, T. et al. (1993) Isolation and characterization of a yeast gene that is homologous with a meiosis-specific cDNA from a plant, Mol. Gen. Genet. 237, 225–232 50 Taylor, R.M. et al. (1998) Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue, Plant J. 14, 75–81 51 Wei, W. et al. (1997) A novel nucleic acid helicase gene identified by promote trapping in Arabidopsis, Plant J. 11, 1307–1314 52 Harlow, G.R. et al. (1994) Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation, Plant Cell 6, 227–235
Vera Gorbunova and Avraham A. Levy* are at the Plant Sciences Dept, Weizmann Institute of Science, Rehovot 76100, Israel. *Author for correspondence (tel 1972 89342734; fax 1972 89344181; e-mail [email protected]).
Ethylene perception and signaling: an evolutionary perspective Anthony B. Bleecker Ethylene signal transduction, as revealed by studies in Arabidopsis, provides an interesting example of how information-processing systems have evolved in plants. The ethylene signal is perceived by a family of receptors composed of structural elements that are characteristic of bacterial signaling proteins. In plants, these receptors transmit the signal by interacting with proteins that are eukaryotic in origin. The ethylene sensor domain of the receptors forms a membrane-associated structure that uses a copper cofactor to bind ethylene. This novel protein motif appears to have originated early in the evolution of photosynthetic organisms.
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n biological systems, responses to chemical signals are generally initiated by ligand-induced conformational changes in a target receptor. The information implicit in these structural changes is propagated, amplified and translated into various forms through a series of events referred to as signal transduction. The steps along a signal-transduction pathway can be mediated by changes in protein–protein interactions, regulation of catalytic activities, activation of gated channels and various combinations of all three. Whereas individual signaling pathways are unique, the domains and motifs that are employed by proteins mediating steps in any one pathway can be common to many signaling pathways. In fact, they are found in diverse lineages of organisms, indicating that many of the basic mechanisms for informationprocessing evolved early in the evolution of life on earth. As a consequence, reconstructing specific signal-transduction pathways is becoming as much an exercise in piecing together recognizable signaling modules as it is in discovering novel mechanisms. This has certainly been the case for signaling by the plant hormone ethylene. The history of ethylene research is a venerable one; ethylene being the first chemically identified endogenous regulator of plant growth and development. More recently, ethylene has been distinguished as the chemical signal about which the most is known concerning the molecular basis for perception and early signal transduction. This recent progress has been driven by the mutational analysis of ethylene-
regulated seedling growth in Arabidopsis2, and by the increasing facility with which Arabidopsis genes can be cloned based on mutant phenotype. The derived amino acid sequences of genes involved in ethylene signaling have invariably provided clues to biochemical function through their homology to signaling components in other systems. In this review, I will summarize some of the recent developments that have significantly increased our understanding of how the ethylene signal is perceived and processed in plant cells. Along the way, I will consider how the molecular details of ethylene signaling are also providing interesting hints concerning the evolutionary origin of this complex regulatory system. The ethylene receptor: conservation of structural elements
Mutations in the ETR1 gene from Arabidopsis confer global insensitivity to ethylene3. Positional cloning of the gene, and subsequent sequence analysis, led to the discovery that the derived protein had several of the structural features of the large family of environmental-sensor proteins from bacteria4. These so-called two-component systems often involve as a first component, a sensor composed of a signal-input domain and a catalytic (histidine kinase) transmitter domain5. Autophosphorylation of a conserved histidine in the transmitter domain is followed by phosphate transfer to a conserved aspartate residue in the receiver domain of the second component in the system, the response regulator.
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trends in plant science reviews domain of ETR1 contains all the conserved residues essenTable 1. Modular structure of signaling proteins from eukaryotic and tial for histidine-kinase activity prokaryotic organisms in the homologous bacterial transmitter domains4, and is Kingdom Species Protein Structurea Function Ref. capable of autophosphorylating on the conserved histiLight sensing 13 HNGFG D Plantae Arabidopsis ETR1 dine when expressed in yeast7. The presence of a C-terminal C GFg Ethylene sensing 4 Plantae Arabidopsis PHYA receiver domain in ETR1 is also a feature of some bacHNGFG D Cytokinin signaling? 30 Plantae Arabidopsis CKI1 terial systems, where it might function as a competing subHNGFG Osmoregulation 8 D Fungi Saccharomyces SIn1p strate for phosphotransfer, or as a relay station in a series of C Ethylene sensing? 9 Eubacteria Synechocystis sIr1212 phosphotransfer steps8. Light sensing 13 C Eubacteria Synechocystis Cph1, Rcp1 HNGFG D Additional evidence of a bacterial origin for the ethylSynthesis of cAMP 10 Eubacteria Anabaena CyaB2 ene receptor was provided by the discovery that an open Oxygen sensing 12 Eubacteria Rhizobium FixL, FixJ HN FG D reading frame, designated slr1212, in the genome of the a cyanobacterium Synechocystis Structural domains are represented as follows: three black rectangles, ethylene-binding domain; white rectangle, membrane-spanning domain; long black rectangle, histidine kinase domain (essential subdomains are indicated by strain 6803 contains a doupper-case letters where complete and lower case letters where incomplete conservation is observed); dark-blue hexamain with homology to the gon, receiver domain; light-blue diamond, GAF domain; light-blue diamond with a ‘C’ inside, GAF-like chromophoreethylene-binding domain of binding domain; small green circle, PAS domain; large purple elipse, adenylate cyclase catalytic domain; yellow lozenge ETR1 (Fig. 1). Ethylenewith a PAS domain in the middle, heme-binding domain. binding assays on Synechocystis cells verified the presence of high-affinity bindThe basic two-component architecture is conserved in the ETR1 ing sites for ethylene. Synechocystis strains, in which the slr1212 protein (Table 1). The hydrophobic domain at the N-terminus gene was disrupted by homologous recombination, lacked ethylshows high-affinity ethylene-binding activity when expressed in ene-binding activity, indicating that slr1212 coded for a functional yeast6, thus qualifying it as a signal-input module. The transmitter ethylene-binding protein9. The biological function of slr1212 remains obscure given that Synechocystis makes no detectable ethylene and V A has no known response to applied ethylene. The slr1212 protein has no histislr1212 A N S Y I P H G H C Y L W Q T P L V W L H V S A D F F T A I A Y Y S I P L T L L 53 dine-kinase module, but does contain ETR1 M E V C N C I E P Q W P A D E L L M K Y Q Y I S D F F I A I A Y F S I P L E L I 40 both GAF (Ref. 10) and PAS (Ref. 11) ERS1 M E S C D C F E T H V N Q D D L L V K Y Q Y I S D A L I A L A Y F S I P L E L I 40 domains (Table 1), features that are nr M E S C D C I E A L L P T G D L L V K Y Q Y L S D F F I A V A Y F S I P L E L I 40 common to several signaling proteins. PAS domains are conserved sequence F/A Y /S A motifs that are thought to play a role in protein–protein interactions associated slr1212 Y F L R K R Q D I P F P N II F L F S T F I L C C G T S H F F D I I T L W Y — — 93 with signal transduction. These domains ETR1 Y F V K K S A V F P Y R W V L V Q F G A F I V L C G A T H L I N LW T F T T H S 80 are found in such sensors as the FixL ERS1 Y F V Q K S A F F P Y K W V L M Q F G A F I I L C G A T H F I N LW M F F M H S 80 oxygen-sensing two-component system nr Y F V H K S A C F P Y R W V L M Q F G A F I V L C G A T H F I S LW T F F M H S 80 in nitrogen-fixing bacteria12 and in phytochromes, where genetic evidence A S T A A indicates that the PAS domains are essential for downstream signaling13. slr1212 — P I Y W I S G T V K A S M A I V S II T V F E L IQ I V P N A L N L K S P T E 133 GAF domains are sequence motifs that ETR1 R T V A L V M T T A K V L T A V V S C A T A L M L V H I IP D L L S V K T R E L 120 have been associated with cyclicERS1 K A V A I V M T I A K V S C A V V S C A T A L M L V H I IP D L L S V K N R E L 120 nucleotide binding sites in a variety of nr K T V A V V M T I S K M L T A A V S C I T A L M L V H I IP D L L S V K T R E L 120 proteins10. They are also associated with the site of chromophore attachment in Fig. 1. Sequence alignment of ethylene-binding domains from the cyanobacterial slr1212 gene the phytochromes, indicating a diversiand ETR1-related ethylene receptors from plants. Amino acid alignments are of the three hydrofication of function over evolutionary phobic subdomains of slr1212 from Synechocystis, ETR1 (Ref. 4) and ERS (Ref. 31) from history. The functional significance of Arabidopsis and nr from tomato (Ref. 32). The lines drawn underneath the sequences indicate the the GAF-containing domain in the ETR1 hydrophobic subdomains. Amino acid substitutions that either disrupt (black), reduce (white) or protein is unknown. It lacks the conserved have no negative effect (gray) on ethylene binding in the yeast-expressed ETR1 protein are indicated by arrows. Alignments were performed using the Clustal method (DNASTAR). cysteine that is essential for chromophore attachment in the phytochromes. 270
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Fig. 2. The structural characteristics of members of the ETR1 family of ethylene receptors from Arabidopsis. For all members of the family, homology extends from the ethylene-binding domain through the histidine kinase-related transmitter domain (long, black rectangle). Where present, conserved subdomains characteristic of functional histidine kinases are indicated by the letters H, N, G, F and G. Based on sequence similarities and defining structural characteristics, the family can be divided into the ETR1 subfamily (a) and the ETR2 subfamily (b). Amino acid substitutions that individually confer dominant insensitivity throughout the plant are indicated. The hydrophobic N-terminal extension that is characteristic of the ETR2 subfamily has the earmarks of a membrane-targeting signal sequence, although such a sequence does not appear to be necessary for membrane association of the ETR1-subfamily members that lack this extension. Additional structural domains present in the ETR1 family of ethylene receptors are indicated as follows: three black rectangles, ethylene-binding domain; gray diamond, GAF domain; gray hexagon, receiver domain.
Interestingly, the GAF domain in Synechocystis slr1212 does contain the conserved cysteine, opening the possibility that this protein is capable of sensing both ethylene and light signals. The recurring presence of common protein motifs in different classes of signaling systems (Table 1) supports the idea from current evolutionary theory that novel systems might arise from the reshuffling of a few basic functional modules. The presence of these motifs across a range of prokaryotes and eukaryotes suggests an ancient ancestry for these signaling modules. The fact that all three higher-plant signaling proteins (ETR1, PHY A and CKI1) are composed of structural elements that are found in the Synechocystis genome is particularly intriguing. Synechocystis is thought to share a common ancestor with the endosymbiont that evolved into the higher-plant chloroplast14. It is a reasonable possibility that at least some of the inherited modules were recruited by the host genome as genetic material that migrated from endosymbiont to host during evolution. This is a particularly plausible scenario for the ethylene-binding domain, which has not been identified in any other bacterial, fungal or animal genomes sequenced to date.
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Fig. 3. Phylogenetic relationships between ETR1-family receptors from Arabidopsis (gray bold) and tomato (black bold) based on amino acid sequence divergence in the ethylene-binding domains (gray bar). The Synechocystis sequence was defined as the outgroup. Sequences were aligned using the Clustal method (DNASTAR). Structural domains are represented as: three black rectangles, ethylenebinding domain; gray spheres, PAS domain; gray diamond, GAF domain; gray diamond with a ‘C’ inside, GAF-like chromophorebinding domain; long, black rectangle, histidine-kinase domain; gray hexagon, receiver domain. Evolution of the ETR receptor family
The Arabidopsis ETR1 gene represents a small gene family in Arabidopsis that is currently composed of five members15. The family members are tied together by common structural elements in the encoded protein and by the finding that amino acid substitutions, in the input domain of any family member, confer dominant insensitivity to ethylene throughout the plant15 (Fig. 2). The receptor family can be divided into two subfamilies based on structural similarities. The ETR1-like subfamily, containing ETR1 and ERS1, is characterized by having three hydrophobic subdomains at the N-terminus and a conserved histidine-kinase domain. Whereas,the ETR2-like subfamily, including ETR2, EIN4 and ERS2, show an additional hydrophobic extension at the Nterminus and carry degenerate histidine-kinase domains that lack one or more elements considered necessary for catalytic activity. In Arabidopsis, one member of each subfamily lacks the C-terminal receiver domain that is characteristic of the other members. Sequence alignments of the ethylene-binding domains of the five Arabidopsis proteins and their presumptive tomato orthologs16 indicate that the degree of divergence in the binding domain correlates with the other structural differences in the family members (Fig. 3). A gene duplication event in an ancestor common to Arabidopsis and tomato could account for the founding of the two receptor subfamilies. Additional duplications later in evolution appear to have occurred in both receptor subfamilies. Receptor sequences from more divergent plant lineages will be required to determine how early in the evolution of land plants these family members diverged. Ethylene responses have been documented in members of most of the major plant lineages1, indicating that some form of the ethylene receptor was present as plants colonized land. If the ancestral form of the receptor July 1999, Vol. 4, No. 7
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Fig. 4. A structural model for the ethylene-binding site of the ethylene receptor. The three hydrophobic subdomains can be modeled as membrane-spanning helices in the topology depicted as a helical net model. ETR1 monomers are covalently linked via disulfide bonds at the extracytoplasmic N-terminus to form a homodimer of ETR1 subunits (Ref. 33). Amino acid residues within this structure that are important for binding activity have been identified by conservation between the plant and the Synechocystis residues (outlined in blue), and by amino acid substitutions that reduce (yellow), abolish (red) or do not disrupt (green) ethylene binding in the yeast-expressed ETR1 protein (Ref. 25). For the second and third helices, conserved residues tend to align along a single face of the modeled helix. Within the conserved face of the second helix, a cysteine residue and a histidine residue are both essential for binding and, in the case of the cysteine, for copper association (Ref. 9). These two residues are probably providing ligands that coordinate the copper cofactor in the binding site.
included a C-terminal receiver domain, it would appear that the loss of this domain by ERS1 and ERS2 occurred independently, later in the evolution of the two receptor subfamilies. The conservation of a functional histidine-kinase domain in the ETR1-subfamily and lack thereof in the ETR2-subfamily implies some divergence in function for the presumptive transmitter domains of the two subfamilies. The lack of conservation in the ETR2-subfamily could be because of genetic drift resulting from a lack of selection for the kinase function, or could be the result of selection for an alternative function. The evolution of plant phytochromes provides an interesting parallel. The bacterial forms of phytochrome appear to operate according to the two-component paradigm, whereas the eukaryotic forms lack conservation in the histidine-kinase domain, and appear to signal downstream through an adjacent PAS domain13. Biochemical evidence indicates that plant phytochrome can autophosphorylate on a serine residue, providing a possible mechanism for receptor adaptation. Evolutionary origin of signal transduction components
Several dowstream components involved in ethylene signal processing have been identified genetically in Arabidopsis2. Loss-offunction mutations in the CTR1 gene result in a constitutive ethylene-response phenotype, indicating that the product of this gene negatively regulates the ethylene-response pathways17. Mutations in CTR1 are epistatic to mutations in each of the receptor genes, indicating that CTR1 acts downstream of the receptors. Sequence analysis of CTR1 revealed that the gene codes for a 272
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serine or threonine protein kinase, related to the mammalian RAF kinases that initiate MAP-kinase cascades. MAP-kinase cascades are often involved in regulating transcription factors18, and, in the case of ethylene signaling, the EIN3 family of transcription factors act genetically downstream of CTR1 and are required for ethylene signaling19. The EIN3-like transcription factors apparently initiate a transcriptional-activation cascade involving members of a large, plant-specific gene family of transcriptional regulators, referred to as EREBPs (Ref. 20). The recruitment of a presumptive MAP-kinase cascade in ethylene signaling represents the coupling of a primarily prokaryotic signaling system with an exclusively eukaryotic signal processing system. An interesting parallel is the two-component based osmosensing system in yeast (Table 1), where a histidine-kinaseinitiated phosphorelay regulates a MAP-kinase cascade through the interaction of a response regulator with a RAF-like kinase8. However, the ethylene system might not operate through the same mechanism as the yeast system. No intermediate components between the ethylene receptors and the CTR1 kinase have been identified genetically or biochemically. Rather, biochemical and yeast two-hybrid studies indicate that CTR1 interacts directly with the transmitter domains of the ethylene receptors21. This raises the possibility that signaling between receptors and CTR1 might not even directly involve phosphotransfer. In any case, the observed differences in component interactions point to an independent origin for the coupling of the two-component sensor and the MAP-kinase cascade signaling systems for osmosensing in yeast and ethylene sensing in plants, respectively. A unique mechanism is also probably acting in ethylene signaling in the downstream regulation of transcription by the presumptive MAP-kinase cascade. Whereas MAP kinases usually regulate transcription directly by phosphorylating transcription factors18, the product of the EIN2 gene, which is related to the eukaryotic Nramp family of presumptive metal ion transporters22, is required for ethylene signaling and acts genetically between CTR1 and the EIN3 family of transcriptional regulators19. Whether the EIN2 gene product acts indirectly in ethylene signaling by affecting metal homeostasis as other Nramp family members are thought to do in some animal systems22, or whether, alternatively, the EIN2 protein is a family member that has been recruited to function directly in ethylene signaling, such as by regulating a second messenger, remains an open and intriguing question. Biochemical basis of ethylene perception
The utility of ethylene as a signal depends on the ability of cells to monitor the changing concentrations of ethylene and to transduce this information into physiological responses appropriate to the cell type. Because ethylene is physiologically effective in the nM range of concentrations, high-affinity receptors for the hormone are required. Ironically, the simplicity of the ethylene molecule presents a particular challenge for proteinaceous receptors, which generally rely on a complex pattern of contact points between binding site and ligand to achieve a high-affinity interaction. This summing of many weak, noncovalent contact points is simply not an option when the signal consists of two hydrogenated carbons that share a double bond. In the 1960s it was suggested that a transitionmetal cofactor could provide the necessary chemistry for highaffinity interaction based on the known ability of olefins to form stable complexes with transition metals23. This hypothesis was substantiated by the recent demonstration that a copper ion is required for the binding of ethylene to the ETR1 receptor from Arabidopsis9. A biochemical understanding of the interaction of ethylene with its binding site has been provided by the discovery that heterologous expression of the Arabidopsis ETR1 receptor in yeast led to the
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Any model demonstrating how ETR-family receptors signal to downstream effectors must account for several experimental observations: • Similar amino acid substitutions in the ethylene-binding domain of any one of the five known members of the ETR family confer global insensitivity to ethylene that is genetically dominant (Fig. 2). • The majority of these point mutations also disrupt ethylenebinding activity in the yeast-expressed ETR1 protein (Fig. 4). • Loss-of-function mutations in any single receptor family member do not cause measurable effects on ethylene signaling in planta26. • Combining loss-of-function mutations for three or more receptor family members in a single plant results in a constitutiveresponse (Ctr1-like) phenotype26. A single model that can account for the above set of observations is one where ethylene acts as a negative regulator of receptor signaling (Fig. 5). According to this model, receptors would actively signal in the absence of ethylene and this signal would act to suppress ethylene-response pathways. Binding of ethylene would suppress signaling by the receptor and thus release response pathways from inhibition. Biochemical evidence21 supports the possibility that the receptors work by interacting directly with the CTR1 gene product (Fig. 5). The model accounts for both dominant mutants that cause ethylene insensitivity, and the constitutive-response phenotype resulting from combined loss-offunction mutants. An implication of the presented model is that all receptor isoforms signal through the same downstream pathway, so each isoform might contribute quantitatively but not qualitatively to the output signal from the primary response pathway. A second implication
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generation of high-affinity ethylene-binding sites in the yeast cells6. The observed binding activity showed many of the characteristics of ethylene-binding sites that have been measured in plant tissues since the late 1970s (Ref. 24), including a dissociation constant (Kd) for binding in the nM range, a half time release of several hours, and inhibition of binding by a variety of competitive inhibitors of ethylene responses in plant tissues. Further experiments demonstrated that the first 128 amino acids of ETR1, representing the hydrophobic region of the protein, were necessary and sufficient for ethylene-binding activity9. In the course of purifying the ETR1 ethylene-binding activity, it was discovered that the addition of copper ions was required for the recovery of binding activity in yeast extracts. Subsequently, it was shown that copper co-purified with binding protein in stoichiometric amounts9. Both ethylene-binding activity and copurification of copper were eliminated when the etr1-1 mutation, a conversion of Cys65 to Tyr, was introduced into the protein. Of several transition metals tested, only silver ions mimicked the effect of copper. This is consistent with the close chemical similarities of these two ions, and also provides a possible explanation for the inhibitory effects of silver ions on ethylene responses in vivo1. Silver appears to be capable of replacing copper and interacting with ethylene, but not in transducing the signal to downstream effectors. A model for ethylene perception by ETR1 has been suggested25 in which an electron-rich hydrophobic pocket, formed by membrane-spanning helices, coordinates a copper(I) cofactor that interacts directly with ethylene. The binding site has been defined25 by in vitro mutagenesis and by the sequence conservation between the Arabidopsis receptors and the cyanobacterial ethylene-binding domain of slr1212 (Fig 4). Binding of ethylene is presumed to alter the coordination chemistry of the copper, resulting in a conformational change in the binding site that in turn is propagated to the transmitter domains of the ETR1 dimer pair.
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Fig. 5. A model of ethylene signal transduction that accounts for phenotypes of loss-of-function mutants. (a) In the absence of ethylene, wild-type receptors activate CTR1, which, in turn, represses response pathways. (b) Ethylene-binding switches off the receptors, resulting in derepression of response pathways. (c) Gain-of-function mutations in any one receptor isoform lead to dominant insensitivity to ethylene by continuing to activate CTR1 in the presence of ethylene. (d) Null mutations in three or more receptor family members result in reduced activation of CTR1, leading to a constitutive-response phenotype.
is that all members of the family participate, to some degree, in the examined range of ethylene responses throughout the plant, because the dominant mutations in any one member, driven by its own promoter, cause global ethylene insensitivity. However, analysis of mRNA levels for individual receptor isoforms indicates that individual receptor family members are expressed at different levels in different tissues15,27. Therefore, the ratios of family members, in a particular cell type, might influence the dose–response relationships, which can vary considerably for different tissues and responses28. The current model (Fig. 5), even if basically correct, is certainly an oversimplification of the actual mechanism of ethylene signaling by the receptors, and, in fact, reveals an interesting paradox. If continued signaling of one mutant receptor isoform in the presence of ethylene is sufficient to repress ethylene responses (Fig. 5), why do as many as two functional receptor isoforms fail to repress July 1999, Vol. 4, No. 7
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trends in plant science reviews ethylene responses in the absence of ethylene in the triple lossof-function mutants (Fig. 5)? One explanation could be that the point mutations that cause dominant insensitivity lock the mutant receptor in a hyperactive-signaling state, which compensates for lack of signaling from wild-type isoforms. An alternative possibility is that mutant receptors use wild-type receptors to signal CTR1, perhaps through the formation of oligomeric complexes of interacting receptors. A model of this sort has been proposed for two-component-based chemoreceptors in bacteria, where clusters of receptors interact in such a way that the conformational state of one receptor can influence the conformational and, thus, signaling states of adjacent receptors29. Concluding remarks
The emerging picture of ethylene signal transduction in plants is turning out to be as complex and sophisticated as the signaling pathways in mammals. In fact, many of the components are cut from the same cloth, illustrating the parsimony with which evolutionary forces have operated over the long history of life on earth. The ability to sense ethylene appears to have originated early in the evolution of photosynthetic organisms. The biological function of the slr1212 gene in Synechocystis is unknown, but the structural and functional homology of the ethylene-binding site to that of the higher-plant receptors is unequivocal. In principle, utilization of this binding activity as a signal depended on the evolution of a complex signalprocessing system in plants. To this end, components were apparently assembled from functional protein motifs that have their origins deep in the roots of the eubacterial domain (histidine kinases), the eukaryotic domain (MAP kinase cascades), and in more recently evolved superfamilies specific to plants (EREBPs). The adoption of these various classes of modules into a coherent pathway could have been achieved primarily by selection for specific protein–protein and/or protein–DNA interaction domains between components. In addition, the recruitment of genes from superfamilies has provided the potential for crosstalk with other regulatory pathways that use related members of the superfamilies. The resultant networks of interconnected signaling systems allow for the integration of signal inputs from a variety of sources and the mounting of complex sets of responses that are essential to the survival of the plant. Acknowledgements
Thanks to Fernando Rodríguez and Jeff Esch for development of the figures, to Harry Klee for sharing unpublished work, and to my laboratory for useful discussions and review of the manuscript. My research is supported by the Dept of Energy (ER20029.00) and the National Science Foundation (MCB-9603679). References 1 Abeles, F.B., Morgan, P.W. and Saltveit, M.E., Jr (1992) Ethylene in Plant Biology (2nd edn), Academic Press 2 Johnson, P.B. and Ecker, J.R. (1998) The ethylene gas signal transduction pathway, Annu. Rev. Genet. 32, 227–254 3 Bleecker, A.B. et al. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241, 1086–1089 4 Chang, C. et al. (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators, Science 262, 539–544 5 Parkinson, J.S. (1993) Signal transduction schemes of bacteria, Cell 73, 857–871 6 Schaller, G.E. and Bleecker, A.B. (1995) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene, Science 270, 1809–1811 7 Gamble, R.L., Coonfield, M.L. and Schaller, G.E. (1998) Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 95, 7825–7829 8 Wurgler-Murphy, S.M. and Saito, H. (1997) Two-component signal transducers and MAPK cascades, Trends Biochem. Sci. 22, 172–176
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9 Rodríguez, F.I. et al. (1999) A copper cofactor for the ETR1 receptor from Arabidopsis, Science 283, 996–998 10 Aravind, L. and Ponting, C.P. (1998) The GAF domain: an evolutionary link between diverse phototransducing proteins, Trends Biochem. Sci. 22, 458–459 11 Zhulin, I.B., Taylor, B.L. and Dixon, R. (1997) PAS domain S-boxes in Archaea, bacteria, and sensors for oxygen and redox, Trends Biochem. Sci. 22, 331–333 12 Gong, W.M. et al. (1998) Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction, Proc. Natl. Acad. Sci. U. S. A. 95, 15177–15182 13 Elich, T.D. and Chory, J. (1997) Phytochrome: if it looks and smells like a histidine kinase, is it a histidine kinase? Cell 91, 713–716 14 Wilmotte, A. (1994) Molecular evolution and taxonomy of the cyanobacteria, in The Molecular Biology of Cyanobacteria (Bryant, D.A., ed.), pp. 1–25, Kluwer 15 Hua, J. et al. (1998) EIN4 and ERS2 are members of the putative ethylene receptor gene family, Plant Cell 10, 1321–1332 16 Tieman, D. and Klee, H.J. (1999) Differential expression of two novel members of the tomato ethylene receptor family, Plant Physiol. 120, 165–172 17 Kieber, J.J. et al. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427–441 18 Treisman, R. (1996) Regulation of transcription by MAP kinase cascades, Curr. Opin. Cell Biol. 8, 205–215 19 Chao, Q. et al. (1997) Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE INSENSITIVE3 and related proteins, Cell 89, 1133–1144 20 Solano, R. et al. (1998) Nuclear events in ethylene signaling: a transduction cascade mediated by ETHYLENE INSENSITIVE3 and ETHYLENE RESPONSE FACTOR1, Genes Dev. 12, 3703–3714 21 Clark, K.L. et al. (1998) Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors, Proc. Natl. Acad. Sci. U. S. A. 95, 5401–5406 22 Flemming, M.D. and Andrews, N.C. (1998) Mammalian iron transport: an unexpected link between metal homeostasis and host defense, J. Lab. Clin. Med. 132, 464– 468 23 Burg, S.P. and Burg, E.A. (1967) Molecular requirements for the biological activity of ethylene, Plant Physiol. 42, 144–152 24 Bleecker, A.B. (1997) The ethylene binding site of the ETR1 protein, in Biology and Biotechnology of the Plant Hormone Ethylene (Kanellis, A. et al., eds), pp. 63–70, Kluwer 25 Bleecker, A.B. et al. (1998) The ethylene receptor family from Arabidopsis: structure and function, Philos. Trans. R. Soc. London Ser. B 353, 1405–1412 26 Hua, J. and Meyerowitz, E.M. (1998) Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana, Cell 94, 261–271 27 Lashbrook, C.C., Teiman, D.M. and Klee, H.J. (1998) Differential regulation of the tomato ETR gene family throughout plant development, Plant J. 15, 243–252 28 Chen, Q. and Bleecker, A.B. (1995) Analysis of ethylene signal transduction kinetics associated with seedling-growth response and chitinase induction in wild-type and mutant Arabidopsis, Plant Physiol. 108, 597–607 29 Bray, D., Levin, M.D. and Morton-Firth, C.J. (1998) Receptor clustering as a cellular mechanism to control sensitivity, Nature 393, 85–88 30 Kakimoto, T. (1996) CKI1, a histidine kinase homolog implicated in cytokinin signal transduction, Science 274, 982–985 31 Hua, J. et al. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712–1714 32 Wilkinson, J.Q. et al. (1995) An ethylene-inducible component of signal transduction encoded by Never-ripe, Science 270, 1807–1809 33 Schaller, G.E. et al. (1995) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer, J. Biol. Chem. 270, 12526–12530
Anthony B. Bleecker is at the Dept of Botany, 430 Lincoln Drive, University of Wisconsin, Madison, WI 53706, USA (tel 11 608 262 4009; e-mail [email protected]).
49 Kobayashi, T. et al. (1993) Isolation and characterization of a yeast gene that is homologous with a meiosis-specific cDNA from a plant, Mol. Gen. Genet. 237, 225–232 50 Taylor, R.M. et al. (1998) Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue, Plant J. 14, 75–81 51 Wei, W. et al. (1997) A novel nucleic acid helicase gene identified by promote trapping in Arabidopsis, Plant J. 11, 1307–1314 52 Harlow, G.R. et al. (1994) Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation, Plant Cell 6, 227–235
Vera Gorbunova and Avraham A. Levy* are at the Plant Sciences Dept, Weizmann Institute of Science, Rehovot 76100, Israel. *Author for correspondence (tel 1972 89342734; fax 1972 89344181; e-mail [email protected]).
Ethylene perception and signaling: an evolutionary perspective Anthony B. Bleecker Ethylene signal transduction, as revealed by studies in Arabidopsis, provides an interesting example of how information-processing systems have evolved in plants. The ethylene signal is perceived by a family of receptors composed of structural elements that are characteristic of bacterial signaling proteins. In plants, these receptors transmit the signal by interacting with proteins that are eukaryotic in origin. The ethylene sensor domain of the receptors forms a membrane-associated structure that uses a copper cofactor to bind ethylene. This novel protein motif appears to have originated early in the evolution of photosynthetic organisms.
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n biological systems, responses to chemical signals are generally initiated by ligand-induced conformational changes in a target receptor. The information implicit in these structural changes is propagated, amplified and translated into various forms through a series of events referred to as signal transduction. The steps along a signal-transduction pathway can be mediated by changes in protein–protein interactions, regulation of catalytic activities, activation of gated channels and various combinations of all three. Whereas individual signaling pathways are unique, the domains and motifs that are employed by proteins mediating steps in any one pathway can be common to many signaling pathways. In fact, they are found in diverse lineages of organisms, indicating that many of the basic mechanisms for informationprocessing evolved early in the evolution of life on earth. As a consequence, reconstructing specific signal-transduction pathways is becoming as much an exercise in piecing together recognizable signaling modules as it is in discovering novel mechanisms. This has certainly been the case for signaling by the plant hormone ethylene. The history of ethylene research is a venerable one; ethylene being the first chemically identified endogenous regulator of plant growth and development. More recently, ethylene has been distinguished as the chemical signal about which the most is known concerning the molecular basis for perception and early signal transduction. This recent progress has been driven by the mutational analysis of ethylene-
regulated seedling growth in Arabidopsis2, and by the increasing facility with which Arabidopsis genes can be cloned based on mutant phenotype. The derived amino acid sequences of genes involved in ethylene signaling have invariably provided clues to biochemical function through their homology to signaling components in other systems. In this review, I will summarize some of the recent developments that have significantly increased our understanding of how the ethylene signal is perceived and processed in plant cells. Along the way, I will consider how the molecular details of ethylene signaling are also providing interesting hints concerning the evolutionary origin of this complex regulatory system. The ethylene receptor: conservation of structural elements
Mutations in the ETR1 gene from Arabidopsis confer global insensitivity to ethylene3. Positional cloning of the gene, and subsequent sequence analysis, led to the discovery that the derived protein had several of the structural features of the large family of environmental-sensor proteins from bacteria4. These so-called two-component systems often involve as a first component, a sensor composed of a signal-input domain and a catalytic (histidine kinase) transmitter domain5. Autophosphorylation of a conserved histidine in the transmitter domain is followed by phosphate transfer to a conserved aspartate residue in the receiver domain of the second component in the system, the response regulator.
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trends in plant science reviews domain of ETR1 contains all the conserved residues essenTable 1. Modular structure of signaling proteins from eukaryotic and tial for histidine-kinase activity prokaryotic organisms in the homologous bacterial transmitter domains4, and is Kingdom Species Protein Structurea Function Ref. capable of autophosphorylating on the conserved histiLight sensing 13 HNGFG D Plantae Arabidopsis ETR1 dine when expressed in yeast7. The presence of a C-terminal C GFg Ethylene sensing 4 Plantae Arabidopsis PHYA receiver domain in ETR1 is also a feature of some bacHNGFG D Cytokinin signaling? 30 Plantae Arabidopsis CKI1 terial systems, where it might function as a competing subHNGFG Osmoregulation 8 D Fungi Saccharomyces SIn1p strate for phosphotransfer, or as a relay station in a series of C Ethylene sensing? 9 Eubacteria Synechocystis sIr1212 phosphotransfer steps8. Light sensing 13 C Eubacteria Synechocystis Cph1, Rcp1 HNGFG D Additional evidence of a bacterial origin for the ethylSynthesis of cAMP 10 Eubacteria Anabaena CyaB2 ene receptor was provided by the discovery that an open Oxygen sensing 12 Eubacteria Rhizobium FixL, FixJ HN FG D reading frame, designated slr1212, in the genome of the a cyanobacterium Synechocystis Structural domains are represented as follows: three black rectangles, ethylene-binding domain; white rectangle, membrane-spanning domain; long black rectangle, histidine kinase domain (essential subdomains are indicated by strain 6803 contains a doupper-case letters where complete and lower case letters where incomplete conservation is observed); dark-blue hexamain with homology to the gon, receiver domain; light-blue diamond, GAF domain; light-blue diamond with a ‘C’ inside, GAF-like chromophoreethylene-binding domain of binding domain; small green circle, PAS domain; large purple elipse, adenylate cyclase catalytic domain; yellow lozenge ETR1 (Fig. 1). Ethylenewith a PAS domain in the middle, heme-binding domain. binding assays on Synechocystis cells verified the presence of high-affinity bindThe basic two-component architecture is conserved in the ETR1 ing sites for ethylene. Synechocystis strains, in which the slr1212 protein (Table 1). The hydrophobic domain at the N-terminus gene was disrupted by homologous recombination, lacked ethylshows high-affinity ethylene-binding activity when expressed in ene-binding activity, indicating that slr1212 coded for a functional yeast6, thus qualifying it as a signal-input module. The transmitter ethylene-binding protein9. The biological function of slr1212 remains obscure given that Synechocystis makes no detectable ethylene and V A has no known response to applied ethylene. The slr1212 protein has no histislr1212 A N S Y I P H G H C Y L W Q T P L V W L H V S A D F F T A I A Y Y S I P L T L L 53 dine-kinase module, but does contain ETR1 M E V C N C I E P Q W P A D E L L M K Y Q Y I S D F F I A I A Y F S I P L E L I 40 both GAF (Ref. 10) and PAS (Ref. 11) ERS1 M E S C D C F E T H V N Q D D L L V K Y Q Y I S D A L I A L A Y F S I P L E L I 40 domains (Table 1), features that are nr M E S C D C I E A L L P T G D L L V K Y Q Y L S D F F I A V A Y F S I P L E L I 40 common to several signaling proteins. PAS domains are conserved sequence F/A Y /S A motifs that are thought to play a role in protein–protein interactions associated slr1212 Y F L R K R Q D I P F P N II F L F S T F I L C C G T S H F F D I I T L W Y — — 93 with signal transduction. These domains ETR1 Y F V K K S A V F P Y R W V L V Q F G A F I V L C G A T H L I N LW T F T T H S 80 are found in such sensors as the FixL ERS1 Y F V Q K S A F F P Y K W V L M Q F G A F I I L C G A T H F I N LW M F F M H S 80 oxygen-sensing two-component system nr Y F V H K S A C F P Y R W V L M Q F G A F I V L C G A T H F I S LW T F F M H S 80 in nitrogen-fixing bacteria12 and in phytochromes, where genetic evidence A S T A A indicates that the PAS domains are essential for downstream signaling13. slr1212 — P I Y W I S G T V K A S M A I V S II T V F E L IQ I V P N A L N L K S P T E 133 GAF domains are sequence motifs that ETR1 R T V A L V M T T A K V L T A V V S C A T A L M L V H I IP D L L S V K T R E L 120 have been associated with cyclicERS1 K A V A I V M T I A K V S C A V V S C A T A L M L V H I IP D L L S V K N R E L 120 nucleotide binding sites in a variety of nr K T V A V V M T I S K M L T A A V S C I T A L M L V H I IP D L L S V K T R E L 120 proteins10. They are also associated with the site of chromophore attachment in Fig. 1. Sequence alignment of ethylene-binding domains from the cyanobacterial slr1212 gene the phytochromes, indicating a diversiand ETR1-related ethylene receptors from plants. Amino acid alignments are of the three hydrofication of function over evolutionary phobic subdomains of slr1212 from Synechocystis, ETR1 (Ref. 4) and ERS (Ref. 31) from history. The functional significance of Arabidopsis and nr from tomato (Ref. 32). The lines drawn underneath the sequences indicate the the GAF-containing domain in the ETR1 hydrophobic subdomains. Amino acid substitutions that either disrupt (black), reduce (white) or protein is unknown. It lacks the conserved have no negative effect (gray) on ethylene binding in the yeast-expressed ETR1 protein are indicated by arrows. Alignments were performed using the Clustal method (DNASTAR). cysteine that is essential for chromophore attachment in the phytochromes. 270
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Fig. 2. The structural characteristics of members of the ETR1 family of ethylene receptors from Arabidopsis. For all members of the family, homology extends from the ethylene-binding domain through the histidine kinase-related transmitter domain (long, black rectangle). Where present, conserved subdomains characteristic of functional histidine kinases are indicated by the letters H, N, G, F and G. Based on sequence similarities and defining structural characteristics, the family can be divided into the ETR1 subfamily (a) and the ETR2 subfamily (b). Amino acid substitutions that individually confer dominant insensitivity throughout the plant are indicated. The hydrophobic N-terminal extension that is characteristic of the ETR2 subfamily has the earmarks of a membrane-targeting signal sequence, although such a sequence does not appear to be necessary for membrane association of the ETR1-subfamily members that lack this extension. Additional structural domains present in the ETR1 family of ethylene receptors are indicated as follows: three black rectangles, ethylene-binding domain; gray diamond, GAF domain; gray hexagon, receiver domain.
Interestingly, the GAF domain in Synechocystis slr1212 does contain the conserved cysteine, opening the possibility that this protein is capable of sensing both ethylene and light signals. The recurring presence of common protein motifs in different classes of signaling systems (Table 1) supports the idea from current evolutionary theory that novel systems might arise from the reshuffling of a few basic functional modules. The presence of these motifs across a range of prokaryotes and eukaryotes suggests an ancient ancestry for these signaling modules. The fact that all three higher-plant signaling proteins (ETR1, PHY A and CKI1) are composed of structural elements that are found in the Synechocystis genome is particularly intriguing. Synechocystis is thought to share a common ancestor with the endosymbiont that evolved into the higher-plant chloroplast14. It is a reasonable possibility that at least some of the inherited modules were recruited by the host genome as genetic material that migrated from endosymbiont to host during evolution. This is a particularly plausible scenario for the ethylene-binding domain, which has not been identified in any other bacterial, fungal or animal genomes sequenced to date.
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Fig. 3. Phylogenetic relationships between ETR1-family receptors from Arabidopsis (gray bold) and tomato (black bold) based on amino acid sequence divergence in the ethylene-binding domains (gray bar). The Synechocystis sequence was defined as the outgroup. Sequences were aligned using the Clustal method (DNASTAR). Structural domains are represented as: three black rectangles, ethylenebinding domain; gray spheres, PAS domain; gray diamond, GAF domain; gray diamond with a ‘C’ inside, GAF-like chromophorebinding domain; long, black rectangle, histidine-kinase domain; gray hexagon, receiver domain. Evolution of the ETR receptor family
The Arabidopsis ETR1 gene represents a small gene family in Arabidopsis that is currently composed of five members15. The family members are tied together by common structural elements in the encoded protein and by the finding that amino acid substitutions, in the input domain of any family member, confer dominant insensitivity to ethylene throughout the plant15 (Fig. 2). The receptor family can be divided into two subfamilies based on structural similarities. The ETR1-like subfamily, containing ETR1 and ERS1, is characterized by having three hydrophobic subdomains at the N-terminus and a conserved histidine-kinase domain. Whereas,the ETR2-like subfamily, including ETR2, EIN4 and ERS2, show an additional hydrophobic extension at the Nterminus and carry degenerate histidine-kinase domains that lack one or more elements considered necessary for catalytic activity. In Arabidopsis, one member of each subfamily lacks the C-terminal receiver domain that is characteristic of the other members. Sequence alignments of the ethylene-binding domains of the five Arabidopsis proteins and their presumptive tomato orthologs16 indicate that the degree of divergence in the binding domain correlates with the other structural differences in the family members (Fig. 3). A gene duplication event in an ancestor common to Arabidopsis and tomato could account for the founding of the two receptor subfamilies. Additional duplications later in evolution appear to have occurred in both receptor subfamilies. Receptor sequences from more divergent plant lineages will be required to determine how early in the evolution of land plants these family members diverged. Ethylene responses have been documented in members of most of the major plant lineages1, indicating that some form of the ethylene receptor was present as plants colonized land. If the ancestral form of the receptor July 1999, Vol. 4, No. 7
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Fig. 4. A structural model for the ethylene-binding site of the ethylene receptor. The three hydrophobic subdomains can be modeled as membrane-spanning helices in the topology depicted as a helical net model. ETR1 monomers are covalently linked via disulfide bonds at the extracytoplasmic N-terminus to form a homodimer of ETR1 subunits (Ref. 33). Amino acid residues within this structure that are important for binding activity have been identified by conservation between the plant and the Synechocystis residues (outlined in blue), and by amino acid substitutions that reduce (yellow), abolish (red) or do not disrupt (green) ethylene binding in the yeast-expressed ETR1 protein (Ref. 25). For the second and third helices, conserved residues tend to align along a single face of the modeled helix. Within the conserved face of the second helix, a cysteine residue and a histidine residue are both essential for binding and, in the case of the cysteine, for copper association (Ref. 9). These two residues are probably providing ligands that coordinate the copper cofactor in the binding site.
included a C-terminal receiver domain, it would appear that the loss of this domain by ERS1 and ERS2 occurred independently, later in the evolution of the two receptor subfamilies. The conservation of a functional histidine-kinase domain in the ETR1-subfamily and lack thereof in the ETR2-subfamily implies some divergence in function for the presumptive transmitter domains of the two subfamilies. The lack of conservation in the ETR2-subfamily could be because of genetic drift resulting from a lack of selection for the kinase function, or could be the result of selection for an alternative function. The evolution of plant phytochromes provides an interesting parallel. The bacterial forms of phytochrome appear to operate according to the two-component paradigm, whereas the eukaryotic forms lack conservation in the histidine-kinase domain, and appear to signal downstream through an adjacent PAS domain13. Biochemical evidence indicates that plant phytochrome can autophosphorylate on a serine residue, providing a possible mechanism for receptor adaptation. Evolutionary origin of signal transduction components
Several dowstream components involved in ethylene signal processing have been identified genetically in Arabidopsis2. Loss-offunction mutations in the CTR1 gene result in a constitutive ethylene-response phenotype, indicating that the product of this gene negatively regulates the ethylene-response pathways17. Mutations in CTR1 are epistatic to mutations in each of the receptor genes, indicating that CTR1 acts downstream of the receptors. Sequence analysis of CTR1 revealed that the gene codes for a 272
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serine or threonine protein kinase, related to the mammalian RAF kinases that initiate MAP-kinase cascades. MAP-kinase cascades are often involved in regulating transcription factors18, and, in the case of ethylene signaling, the EIN3 family of transcription factors act genetically downstream of CTR1 and are required for ethylene signaling19. The EIN3-like transcription factors apparently initiate a transcriptional-activation cascade involving members of a large, plant-specific gene family of transcriptional regulators, referred to as EREBPs (Ref. 20). The recruitment of a presumptive MAP-kinase cascade in ethylene signaling represents the coupling of a primarily prokaryotic signaling system with an exclusively eukaryotic signal processing system. An interesting parallel is the two-component based osmosensing system in yeast (Table 1), where a histidine-kinaseinitiated phosphorelay regulates a MAP-kinase cascade through the interaction of a response regulator with a RAF-like kinase8. However, the ethylene system might not operate through the same mechanism as the yeast system. No intermediate components between the ethylene receptors and the CTR1 kinase have been identified genetically or biochemically. Rather, biochemical and yeast two-hybrid studies indicate that CTR1 interacts directly with the transmitter domains of the ethylene receptors21. This raises the possibility that signaling between receptors and CTR1 might not even directly involve phosphotransfer. In any case, the observed differences in component interactions point to an independent origin for the coupling of the two-component sensor and the MAP-kinase cascade signaling systems for osmosensing in yeast and ethylene sensing in plants, respectively. A unique mechanism is also probably acting in ethylene signaling in the downstream regulation of transcription by the presumptive MAP-kinase cascade. Whereas MAP kinases usually regulate transcription directly by phosphorylating transcription factors18, the product of the EIN2 gene, which is related to the eukaryotic Nramp family of presumptive metal ion transporters22, is required for ethylene signaling and acts genetically between CTR1 and the EIN3 family of transcriptional regulators19. Whether the EIN2 gene product acts indirectly in ethylene signaling by affecting metal homeostasis as other Nramp family members are thought to do in some animal systems22, or whether, alternatively, the EIN2 protein is a family member that has been recruited to function directly in ethylene signaling, such as by regulating a second messenger, remains an open and intriguing question. Biochemical basis of ethylene perception
The utility of ethylene as a signal depends on the ability of cells to monitor the changing concentrations of ethylene and to transduce this information into physiological responses appropriate to the cell type. Because ethylene is physiologically effective in the nM range of concentrations, high-affinity receptors for the hormone are required. Ironically, the simplicity of the ethylene molecule presents a particular challenge for proteinaceous receptors, which generally rely on a complex pattern of contact points between binding site and ligand to achieve a high-affinity interaction. This summing of many weak, noncovalent contact points is simply not an option when the signal consists of two hydrogenated carbons that share a double bond. In the 1960s it was suggested that a transitionmetal cofactor could provide the necessary chemistry for highaffinity interaction based on the known ability of olefins to form stable complexes with transition metals23. This hypothesis was substantiated by the recent demonstration that a copper ion is required for the binding of ethylene to the ETR1 receptor from Arabidopsis9. A biochemical understanding of the interaction of ethylene with its binding site has been provided by the discovery that heterologous expression of the Arabidopsis ETR1 receptor in yeast led to the
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Any model demonstrating how ETR-family receptors signal to downstream effectors must account for several experimental observations: • Similar amino acid substitutions in the ethylene-binding domain of any one of the five known members of the ETR family confer global insensitivity to ethylene that is genetically dominant (Fig. 2). • The majority of these point mutations also disrupt ethylenebinding activity in the yeast-expressed ETR1 protein (Fig. 4). • Loss-of-function mutations in any single receptor family member do not cause measurable effects on ethylene signaling in planta26. • Combining loss-of-function mutations for three or more receptor family members in a single plant results in a constitutiveresponse (Ctr1-like) phenotype26. A single model that can account for the above set of observations is one where ethylene acts as a negative regulator of receptor signaling (Fig. 5). According to this model, receptors would actively signal in the absence of ethylene and this signal would act to suppress ethylene-response pathways. Binding of ethylene would suppress signaling by the receptor and thus release response pathways from inhibition. Biochemical evidence21 supports the possibility that the receptors work by interacting directly with the CTR1 gene product (Fig. 5). The model accounts for both dominant mutants that cause ethylene insensitivity, and the constitutive-response phenotype resulting from combined loss-offunction mutants. An implication of the presented model is that all receptor isoforms signal through the same downstream pathway, so each isoform might contribute quantitatively but not qualitatively to the output signal from the primary response pathway. A second implication
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generation of high-affinity ethylene-binding sites in the yeast cells6. The observed binding activity showed many of the characteristics of ethylene-binding sites that have been measured in plant tissues since the late 1970s (Ref. 24), including a dissociation constant (Kd) for binding in the nM range, a half time release of several hours, and inhibition of binding by a variety of competitive inhibitors of ethylene responses in plant tissues. Further experiments demonstrated that the first 128 amino acids of ETR1, representing the hydrophobic region of the protein, were necessary and sufficient for ethylene-binding activity9. In the course of purifying the ETR1 ethylene-binding activity, it was discovered that the addition of copper ions was required for the recovery of binding activity in yeast extracts. Subsequently, it was shown that copper co-purified with binding protein in stoichiometric amounts9. Both ethylene-binding activity and copurification of copper were eliminated when the etr1-1 mutation, a conversion of Cys65 to Tyr, was introduced into the protein. Of several transition metals tested, only silver ions mimicked the effect of copper. This is consistent with the close chemical similarities of these two ions, and also provides a possible explanation for the inhibitory effects of silver ions on ethylene responses in vivo1. Silver appears to be capable of replacing copper and interacting with ethylene, but not in transducing the signal to downstream effectors. A model for ethylene perception by ETR1 has been suggested25 in which an electron-rich hydrophobic pocket, formed by membrane-spanning helices, coordinates a copper(I) cofactor that interacts directly with ethylene. The binding site has been defined25 by in vitro mutagenesis and by the sequence conservation between the Arabidopsis receptors and the cyanobacterial ethylene-binding domain of slr1212 (Fig 4). Binding of ethylene is presumed to alter the coordination chemistry of the copper, resulting in a conformational change in the binding site that in turn is propagated to the transmitter domains of the ETR1 dimer pair.
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Fig. 5. A model of ethylene signal transduction that accounts for phenotypes of loss-of-function mutants. (a) In the absence of ethylene, wild-type receptors activate CTR1, which, in turn, represses response pathways. (b) Ethylene-binding switches off the receptors, resulting in derepression of response pathways. (c) Gain-of-function mutations in any one receptor isoform lead to dominant insensitivity to ethylene by continuing to activate CTR1 in the presence of ethylene. (d) Null mutations in three or more receptor family members result in reduced activation of CTR1, leading to a constitutive-response phenotype.
is that all members of the family participate, to some degree, in the examined range of ethylene responses throughout the plant, because the dominant mutations in any one member, driven by its own promoter, cause global ethylene insensitivity. However, analysis of mRNA levels for individual receptor isoforms indicates that individual receptor family members are expressed at different levels in different tissues15,27. Therefore, the ratios of family members, in a particular cell type, might influence the dose–response relationships, which can vary considerably for different tissues and responses28. The current model (Fig. 5), even if basically correct, is certainly an oversimplification of the actual mechanism of ethylene signaling by the receptors, and, in fact, reveals an interesting paradox. If continued signaling of one mutant receptor isoform in the presence of ethylene is sufficient to repress ethylene responses (Fig. 5), why do as many as two functional receptor isoforms fail to repress July 1999, Vol. 4, No. 7
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trends in plant science reviews ethylene responses in the absence of ethylene in the triple lossof-function mutants (Fig. 5)? One explanation could be that the point mutations that cause dominant insensitivity lock the mutant receptor in a hyperactive-signaling state, which compensates for lack of signaling from wild-type isoforms. An alternative possibility is that mutant receptors use wild-type receptors to signal CTR1, perhaps through the formation of oligomeric complexes of interacting receptors. A model of this sort has been proposed for two-component-based chemoreceptors in bacteria, where clusters of receptors interact in such a way that the conformational state of one receptor can influence the conformational and, thus, signaling states of adjacent receptors29. Concluding remarks
The emerging picture of ethylene signal transduction in plants is turning out to be as complex and sophisticated as the signaling pathways in mammals. In fact, many of the components are cut from the same cloth, illustrating the parsimony with which evolutionary forces have operated over the long history of life on earth. The ability to sense ethylene appears to have originated early in the evolution of photosynthetic organisms. The biological function of the slr1212 gene in Synechocystis is unknown, but the structural and functional homology of the ethylene-binding site to that of the higher-plant receptors is unequivocal. In principle, utilization of this binding activity as a signal depended on the evolution of a complex signalprocessing system in plants. To this end, components were apparently assembled from functional protein motifs that have their origins deep in the roots of the eubacterial domain (histidine kinases), the eukaryotic domain (MAP kinase cascades), and in more recently evolved superfamilies specific to plants (EREBPs). The adoption of these various classes of modules into a coherent pathway could have been achieved primarily by selection for specific protein–protein and/or protein–DNA interaction domains between components. In addition, the recruitment of genes from superfamilies has provided the potential for crosstalk with other regulatory pathways that use related members of the superfamilies. The resultant networks of interconnected signaling systems allow for the integration of signal inputs from a variety of sources and the mounting of complex sets of responses that are essential to the survival of the plant. Acknowledgements
Thanks to Fernando Rodríguez and Jeff Esch for development of the figures, to Harry Klee for sharing unpublished work, and to my laboratory for useful discussions and review of the manuscript. My research is supported by the Dept of Energy (ER20029.00) and the National Science Foundation (MCB-9603679). References 1 Abeles, F.B., Morgan, P.W. and Saltveit, M.E., Jr (1992) Ethylene in Plant Biology (2nd edn), Academic Press 2 Johnson, P.B. and Ecker, J.R. (1998) The ethylene gas signal transduction pathway, Annu. Rev. Genet. 32, 227–254 3 Bleecker, A.B. et al. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241, 1086–1089 4 Chang, C. et al. (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators, Science 262, 539–544 5 Parkinson, J.S. (1993) Signal transduction schemes of bacteria, Cell 73, 857–871 6 Schaller, G.E. and Bleecker, A.B. (1995) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene, Science 270, 1809–1811 7 Gamble, R.L., Coonfield, M.L. and Schaller, G.E. (1998) Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis, Proc. Natl. Acad. Sci. U. S. A. 95, 7825–7829 8 Wurgler-Murphy, S.M. and Saito, H. (1997) Two-component signal transducers and MAPK cascades, Trends Biochem. Sci. 22, 172–176
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Anthony B. Bleecker is at the Dept of Botany, 430 Lincoln Drive, University of Wisconsin, Madison, WI 53706, USA (tel 11 608 262 4009; e-mail [email protected]).