Ethylene gas: perception, signaling and response

393

Ethylene gas: perception, signaling and response Roberto Solano and Joseph R Ecker

During the last decade a genetic approach based on the

Arabidopsis ‘triple response’ to the hormone ethylene has

allowed the identification of numerous components of the

signal transduction pathway. Cloning of the genes and

biochemical analysis of the proteins that they encode are

uncovering the molecular mechanisms that allow a plant cell to

perceive and respond to this gaseous regulator of plant

growth/stress responses.

Addresses Department of Biology, Plant Science Institute,University of Pennsylvania. Philadelphia, Pennsylvania 19104-6018; e-mail: [email protected]; [email protected]

Current Opinion in Plant Biology 1998, 1:393-398

http://biomednet.com/elecref/1369526600100393

0 Current Biology Ltd ISSN 1369-5266

Abbreviations AIN ACC(1 -aminocyclopropane-1 -carboxilic acid) insensitive CTR constitutive triple response EIL Ein3-like EIN ethylene insensitive EREBP ethylene response element binding protein ERS ethylene response sensor ETI ethylene insensitive ET0 ethylene overproducer ETR ethylene resistant/ethylene receptor MAPK mitogen-activated protein kinase MEK MAPK kinase

Introduction Ethylene is a phytohormone affecting virtually all stages of plant development. During the course of a plant’s life cycle, ethylene affects seed germination, cell elongation, cell fate, sex determination, fruit ripening, senescence and abscission. Ethylene is also a key regulator in the response to biotic and abiotic stresses [ l-4,5’-8’1. The regulation of all these processes by ethylene depends on the ability of plant cells to perceive the hormone, transduce the signal from the membrane to the nucleus, and activate the expression of specific effector genes involved in the various responses to the hormone.

Seedlings of dicot)rledoneous plants grown in the dark in the presence of ethylene undergo dramatic morphological changes collectively known as the ‘triple response’. These effects were first noticed in pea seedlings [9,10] and in Arahi&p.& include the inhibition of hypocotyl and root elongation, radial swelling of hypocotyl and root cells, and the exaggeration of the apical hook [ll]. The triple response may represent a stress-induced adaptation that allows seedlings to penetrate the soil without damage to the apical meristem [ 121. During the last decade, a genetic approach based on these dramatic morphological changes induced by ethylene in &-nl’/i&sis seedlings has allowed

the identification of several classes of mutants impaired in the response to the hormone. Mutants that display a ‘constitutive’ triple response phenotype may result either from ethylene overproduction (etol, eto2 and efo.?), or constitutive activation of the pathway (ctrl). Insensitive mutants are defective in their ability to perceive or respond to ethylene and include etrl, etr2, ein2, ein3 (Figure l), eZn4, ein5Jain1, einf, ein7 and eti mutants [11,13,14’]. Genetic analysis of these mutants has revealed that they act in a linear pathway [15]. Molecular analysis to elucidate the biochemical function of the proteins identified by these mutations is begin- ning to uncover the components that participate in the perception, signaling and ethylene-mediated transcriptional regulation. Recent results have provided a framework to understand, at the molecular level, the mechanisms that underlie a plant’s responses to this gaseous plant growth/stress response hormone.

In the current view, ethylene is perceived at the plasma membrane by a family of ethylene receptors that includes five members: ethylene resistant/ethylene receptor (ETR)l, ETRZ, ethylene response sensor (ERS)l, ERSZ and ethylene insensitive (EIN)4 [14’,16,17,18’]. From the membrane, the signal is transduced to the nucleus through a series of proteins that include constitutive response (CTR)l, EINZ, EINS, EIN6 and EIN7. Among these, only the cloning of tn-1 has been reported to date [19]. In the nucleus, EIN3 and most likely other members of the EIN3/EIN3-like (EIL) family of positive regulatory proteins, initiate the events that will lead to the expression of effector genes involved in the diversity of responses to the hormone.

In this review we will briefly summarize our current view of the ethylene pathway, focusing on the recent findings on the hormone perception, signaling events and ethylene-mediated transcription.

Ethylene perception: the family of receptors The ethylene receptor family in Arabkz’opsis consists of five members: ETRl, ETRZ, EIN4, ERSl and ERSZ [14’,16,17,18’]. All of them have been implicated in ethylene signaling by dominant missense mutations that confer ethylene insensitivity to the plant._elrZ and em4 were the first members of the family isolated by the ‘triple response’ screen [ lS,ZO]. Recently, dominant mutations in the ETRZ and ERSZ genes have also been obtained from the same type of screen ([14-l; JM Alonso, JR Ecker, Abstract 385, 9th International Conference on At-abidopsis Research, 24-28 June 1998, Madison WI). In addition, mutations that confer dominant ethylene insensitivity to ETRI have been introduced into ERSl and ERS2, and the resulting transgenic plants also show an ethylene insensitive phenotype [17,18’]. r;trl is epistatic to all mutants in members of this family and thus, prior to their

394 Cell signalling and gene regulation

Figure 1

Constitutive activation of the ethylene signaling pathway by EIN3 in Arabidopsis. Compared to that grown in air (seedling on the right), a dark-grown wild-type Col-0 Arabidopsis plant exhibits a radial swelling of the hypocotyl, an exaggeration in the curvature of the apical hook, and an inhibition of cell elongation in the hypocotyl and root in the presence of ethylene. Overexpression of EIN3 (seedling on the left) in the wild-type Col-0 background leads to a constitutive ethylene response in the absence of the hormone, mimicking the phenotype of a wild-type Col-0 seedlings grown in the presence of ethylene.

molecular characterization, genetic studies suggested that these genes acted early in the ethylene signaling pathway ([14’,1.5,17,18’]; JM Alonso, JR Ecker, Abstract 38.5, 9th International Conference on AmhiiQsis Research, 24-28 June 1998, Madison WI).

ETRZ was the first member cloned, using a positional approach [16]. All other members of the family were isolated due to their sequence homology to ETRl (ERSI and ETRZ; [14’,17]) or to ETRZ (EZN4 aKd ERS,7; [18’]). Sequence analysis of the ETRl gene uncovered its similarity to two-component histidine-kinase regulators that are sensors and transducers of environmental signals in bacteria [Zl]. Since then, other two-component sensors have been found in eukaryotes, including Arahidopsis [Z-28]. Two component receptors consist of a sensor protein with a histidine autokinase domain and a response regulator protein. Activation of the histidine-kinase in the sensor protein promotes autophosphorylation of the histidine

residue and a subsequent transfer of the phosphoryl group to an aspartate residue in the receiver domain of the response regulator [Zl]. In ETRl, as well as in ETRZ and EIN4, the sensor and the response regulator components are present in the same protein. ERSl and ERSZ, however, lack the response regulator, suggesting that, as in the case of bacteria, this second component may also exist in plants as an independent protein. In fact, five different response regulators have been recently cloned in Arabidopsis by sequence homology to bacterial response regulators [29’]. Whereas in z&o and in vitro evidence has been provided that they can function in His-Asp phosphotransfer signaling in E. co/i, interactions among any of these proteins and ERSl, ERSZ or the other ethylene receptors have not been reported.

ERSl shares a high degree of sequence similarity with ETRl (72% in the amino-terminus and 64% in the histidine-kinase domain), including all canonical motifs of bacterial histidine protein kinases [17]. EIN4, ETRZ and ERSZ, however, comprise an independent, more divergent class of receptors, in which the hallmarks of bacterial histidine kinases are barely detectable [14’,18’]. Whether or not all of these proteins can function as histidine kinases is not clear, although this activity has been demonstrated in vitro for ETRl [30”]. As the introduction into plants of a mutant version of etrl containing two kind of mutations (mutations that confers dominant ethylene insensitivity, and mutations in an amino acid residue essential for histidine-kinase activity in prototypical ‘two-component’ regulators), still conferred dominant ethylene insensitivity to transgenic plants, the in vivo significance of the histidine kinase domain is unclear ([31], J Hua, E Meyerowitz, Abstract 405, 9th International Conference on Arabidopsis Research, 24-28 June 1998, Madison WI). Homo- or het- ero-dimerization of these receptors, however, may provide, in trans, kinase activity to the ‘killed-kinase’ mutant. Further proof of the in vivo significance of the kinase activity may be obtained by the introduction of various mutant transgenes into the loss-of-function mutant backgrounds (single, double, triple and quadruple) recently obtained by Hua and Meyerowitz [32”].

As mentioned earlier, genetic evidence that these proteins act upstream of CTRl in the ethylene pathway and their similarity to two-component bacterial sensors suggested their roles as ethylene receptors. Supporting biochemical evidence has been provided by ethylene binding experiments. It has been found that etrI mutants bind significantly less ethylene than wild-type plants [ZO]. Furthermore, heterologous expression of ETRl and ERSl in yeast cells allows them to reversibly bind the hormone ([33]; AE Hall, JL Findell, E Schaller, AB Bleecker, abstract 402, 9th International Conference on Amhidopsis Kesearch, 24-28 June 1998, Madison WI).

The-amino terminus of ETRl contains three predicted transmembrane domains that may anchor the protein to

Ethylene gas: perception, signaling and response Solano and Ecker 395

the plasma membrane [16]. In fact, ETRl homodimers have been found to be associated with membranes in tydnSgenic yeast and in Arabidopsis [34]. The unique nature of this region and the fact that all mutations in the dominant alleles of all receptor family members (except for ERSl; JM Alonso, JR Ecker, Abstract 385, 9th International Conference on Arabidopsis Research, 24-28 June 1998, Madison WI) map to this region, suggest a role in ethylene binding. Consistent with this hypothesis, the amino terminus is the most conserved region among the putative receptor proteins; although a fourth transmembrane segment can be predicted in EIN4, ETRZ and ERSZ [14’,18’]. Moreover, mutations in specific Cys residues that are predicted to lie within the transmembrane domain prevent ethylene binding [33]. It has been previously proposed that a transition metal is needed to coordinate the binding of ethylene to the receptors [35,36]. Interestingly, the amino terminal hydrophobic domain of ETRl shares similarity with a protein from Cyanobacteria (SLRlZlZ). This protein may normally function in copper scavenging since a knockout of SLRlZZZ causes increased sensitive to copper (JJ Esch, FI Rodriguez, BM Binder, CE Hetzel, AB Bleecker, 9th International Conference on Arabidopsis Research, 24-28 June 1998, Madison WI). This information together with the finding that the addition of copper to yeast membranes containing ETRl stim- ulates the binding of ethylene, provides strong support to an early suggestion that copper may be the ethylene-coor- dination transition metal in the receptors [35-371.

Nevertheless, only dominant mutations that may result from loss- or gain-of-function, have been found in these proteins, Thus, it has been unclear whether all of these proteins are involved in ethylene perception, and whether they function as positive or negative regulators of the signaling pathway. Recently, key insights into the mechanism through which the receptors transmit the ethylene signal have significantly improved our understanding of the function of the family. Triple and quadruple mutants of loss-of- function mutations in ETRI, ETR2, EIN4 and ERS2, were found to display a strong constitutive ethylene response phenotype, much like curl mutants [32”]. The results ele- gantly demonstrated that these four genes have redundant functions in ethylene perception and negatively regulate ethylene responses. In the absence of ethylene, one or more of these four proteins activates CTRl which, in turn, acts to negatively fegulate downstream ethylene signaling events. Binding of ethylene to the ‘receptors inactivates them, thereby turning off CTRl (Figure 2). Thus, dominant mutations in the receptor proteins most likely lock them in an active state, rendering them unable to bind ethylene or transmit the signal to their cytoplasmic domains.

The question arises as to why all of these genes have been conserved during evolution if they serve redundant functions. The likely answer is that in the plant this redundancy may be only partial and each member of the family may have a specific function. In fact, although the pattern of

expression of these genes is quite ubiquitous, small differ- ences can be observed [14’,18’]. In addition, expression of ETRZ and ERSl is stimulated by ethylene [18’], and dif- ferences in their affinities for ethylene can be expected on the basis of in viva binding assays [37]. The existence of five different ethylene receptors with partially redundant functions may serve as a mechanism to finely regulate the different responses to the hormone under different conditions, and/or to achieve different sensitivities in different tissues. The enhanced expression in particular tissues of enzymes that participate in ethylene biosynthesis may serve a similar purpose. In this context, it has been recently shown that the expression of Ps-AC01 (l-aminocyclopropane-1-carboxylate oxidase) in the apical hook of etiolated pea seedlings is higher in the inner than in the outer side of the hook [38’]. Analysis of the asymmetric distribution of other components of the pathway will provide new clues to understand the effect of the hormone on differential growth responses in particular tissues.

Signal transduction On the basis of epistasis analysis, CTRZ is the initial component of the signaling pathway to act downstream of the receptors. As stated above, CTRl is considered a negative regulator of ethylene signaling since recessive loss-of-function mutations confer constitutive ethylene responses to the plant throughout development [19]. The deduced amino acid sequence of CTRl shares significant similarity with the Raf family of protein kinases, and in fact, its kinase activity has been shown using Arabidopsis daliana MAPK kinase (AtMEK) as a substrate ([19,39]; H Li, Y Huang, J Kieber, abstract 409, 9th International Conference on Arabidopsis Research, 24-28 June 1998, Madison WI). In animals, Raf kinases are Ser-Thr protein kinases that phosphorylate MEKs (MAPK kinases), which in turn phosphorylate MAPKs [40]. In yeast, the osmolarity response pathway represents a paradigm in which a MAPK cascade is linked to a two-component receptor, SLNl [23]. Because of the similarity between CTRl and Raf, and by analogy with the yeast osmolarity response pathway, a MAPK cascade has been proposed to participate in the repression of ethylene responses [19]. In Arabidopsis, nine MAPKs and several MEKs have been identified [41-44], although their roles in ethylene signaling have yet to be demonstrated.

The mechanism of activation of CTRl by the ethylene receptors is still unclear. As in the case of Raf, however, it may require recruitment to the plasma membrane. In fact, interactions between the amino terminus of CTRl, which is predicted to be a regulatory domain on the basis of findings with animal Rafs, and the carboxyl termini of ETRl and ERSl have been demonstrated in vitro and using the yeast two-hybrid system [45”]. In addition, five different 14-3-3 isoforms - ubiquitously expressed proteins that interact with signaling molecules and cell cycle regulators [46] - have been also found to interact with the amino- terminal portion of CTRl in two-hybrid assays, and one of


Figure 2

\ Membrane

\

Nucleus

BASIC-CHITINASE El305 GST2 E4/E8

Current Oplnlon I” Plant &logy Others

The ethylene signaling pathway in Arabidopsis. Shown is a model of the signaling events in response to ethylene that is consistent with genetic, molecular and biochemical analyses. In the absence of the hormone the receptors interact and activate the CTRl protein kinase, which in turn, directly or through a MAPK cascade, negatively regulates downstream signaling events, possibly by repression of the positive acting EIN2 protein. Binding of ethylene to membrane receptors inactivates them and therefore CTRl , which is proposed to be stimulated by receptor interaction, is no longer able to repress EIN2. The nuclear members of the signaling pathway, EIN3/EILs, are positive regulators of ethylene responses, acting downstream of EIN2. This family of proteins (EIN3/EILs) may be involved in the activation of members of the EREBP family of transcription factors, which in turn may regulate the expression of effector genes such as HOOKLESS and basic-chitinase. Active molecules are represented in black and inactive molecules in white. Grey represents molecules that are active in the presence of ethylene and inactive in its absence. Cu, copper ions.

them also interacts with ETRl (W Ding, C Chang, Abstract 392, 9th International Conference on Arabidopsis Research, 24-28 June 1998, Madison WI). ‘I’his suggests that additional factors may be required for the formation of the CTRl-receptor(s) complex in ulvo.

Downstream of the putative MAPK cascade, several genes necessary for ethylene signaling have been described and include EINZ, EINS, EIN6 and EIN7 (reviewed in [11,13,39]). EZNZ and EI1V5 have been already cloned, and EIN6 will be soon (G Roman; S Nourizadeh; R McGrath; personal communications). Molecular characterization of these components will likely provide us with a better understanding of the mechanisms that transduce the ethylene signal from the cytoplasm to the nucleus.

Nuclear members of the signalingpathway: the EINS/EIL family Mutations in EIN3 impair a plant’s response to the hormone through all stages of development. Cloning of the EZN.1 gene revealed that it encodes a novel protein that shares amino acid sequence similarity, conserved structural features and genetic function with four EIN3-LIKE (EIL) proteins ([47”]; R Solano, JR Ecker, unpublished data). Transgenic analysis has shown that EIL,I and EZL2 are able to functionally complement the ein3 mutation,

suggesting their participation in ethylene signaling. High level expression of EIN3 or EZLI in transgenic wild-type or &Z mutant plants conferred constitutive ethylene response phenotypes in all stages of development, indicating their sufficiency for activation of the pathway in the absence of ethylene. The members of this family do not share significant sequence homology with other proteins in databases. Yet sequence analysis indicates that they contain features also found in families of transcription factors, such as acidic and basic domains (including two with predicted a-helical structure) that may represent activation and DNA-binding domains, respectively [47”]. In fact, it has been demonstrated that EIN3 contains signals suff- cient for nuclear localization in transient assays, regardless of plant species or cell type, indicating that this family of proteins may function in the nucleus [47”]. On the basis of these observations, it has been proposed that EIN3 and EILs proteins may represent a new class of transcriptional regulators involved in the activation (or repression) of ethylene-regulated genes [47”].

Likely targets of EIN3/EILs are the ethylene-response- element-binding-protein (EREBP) family of transcription factors. ‘I’hese proteins were originally identified in tobacco by their ability to recognize a conserved ethylene response element (GCC box), and they possess a

Ethylene gas: perception, signaling and response Solano and Ecker 397

DNA-binding domain homologous to that found in the flo- ral homeotic protein APETALAZ (i.e. the AP2 domain;

[11,48,491).

In Arrrhi~@sis, over thirty EREBPs have been identified by several research groups and by the Arzzbidopsls genome sequencing project, and thus they constitute one of the largest families of DNA-binding proteins in plants [~0,51*,52*], R Solano, JR Ecker, unpublished data). This suggests a putative functional redundancy among members of this family that might explain the lack of a loss-of- function mutant in any of these genes. Gain-of-function experiments will be required to demonstrate the role of this family in ethylene signaling.

Conclusions During the last few years the identification and cloning of genes involved in ethylene signaling has considerably improved our understanding of the pathway. It is clear now that the ethylene signal is perceived and transduced through a largely linear pathway that begins at the membrane and proceeds to the nucleus. Acting through a putative CTRl-hlEK-MAP cascade of protein kinases, a family of transmembrane receptors (ETRl, EIN4, ETRZ, ERSl, ERSZ) functions as negative regulators of ethylene signaling events. In the nucleus, a family of positive regulatory proteins (EIN3/EILs) serves to activate transcriptional responses to the hormone upon repression of receptor function by the binding of ethylene.

The identification of new mutants using novel genetic screens and the characterization of additional ethylene signaling genes such as ein2, ein5 and ein6 should clarify our view of the ethylene action pathway. Still, our knowledge is quite limited on the biochemical function of most of the components of the pathway and how they are regulated, providing a significant future challenge.

Acknowledgements The authors wish m apologize to chose ethylene biologists whose research wve could not discuss because of space limitations. \\‘e \+ould all like to thank Jose Alonso, Greg Roman, Robert hlcGrath, Saeid Nourizadch, Jian Hua, and Ellior Rlqerowirz for allowing us tu refer CO unpubli&d rc\ultc. and members of the Ecker laboratory for critical reading of the manuscript. \Ibrk in the Eckcr laborarorr is supported h\ grant? from the National Science Foundarion and the LIcpartment of Energy. R Srrlano has been supported by postdoctoral fcllowthips from the Human Frontiers of Science Program Organization and the Spanish hl~nisrerin de Education r Clcncia.

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Ethylene gas: perception, signaling and response