Naomi Etheridge is a postdoctoral research associate in the Department of Biological Sciences at Dartmouth College. Yi-Feng Chen is a postdoctoral research associate in the Department of Biological Sciences at Dartmouth College. G. Eric Schaller is an associate professor in the Department of Biological Sciences at Dartmouth College.
Naomi Etheridge, Yi-Feng Chen and G. Eric Schaller Date received (in revised form): 6th January 2005
Abstract The plant hormone ethylene regulates growth, development and stress responses. In recent years, various genomic and proteomic approaches have been initiated to understand both the range of ethylene responses in the plant and the mechanism of signal transduction. Transcriptional profiling experiments reveal broad-ranging effects of ethylene upon gene regulation, with up to 7 per cent of the genes examined demonstrating a significant level of response in one study. Both transcriptional and post-transcriptional mechanisms regulate the expression of components within the ethylene signal transduction pathway. The importance of post-transcriptional regulation via the ubiquitin/proteasome-mediated degradation pathway is apparent in studies on the accumulation of ethylene insensitive 3 (EIN3), a key transcription factor in the pathway. Protein complexes also play a role in modulating ethylene signal transduction, with interactions between the ethylene receptors and the Raf-like kinase constitutive triple response-1 (CTR1) being required for ethylene perception at the endoplasmic reticulum. In this paper, recent developments in unravelling the transcriptional and post-transcriptional regulation of the ethylene signalling and response pathways are considered, along with the latest developments in unravelling the biochemical mechanism behind ethylene perception.
INTRODUCTION
G. Eric Schaller Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA Tel: +1 (603) 646 2525 Fax: +1 (603) 646 1347 E-mail:
[email protected] As with other multicellular eukaryotes, hormones are a driving force behind many physiological and developmental processes in plants. The sessile nature of the plant, however, produces unique challenges that animals do not have to face. A plant must survive where it germinates, contending with pathogen attacks, nutrient availability and environmental variations (eg light, temperature, water accessibility). To confront these challenges, plants employ complex signalling networks to detect and respond to external cues from the environment in conjunction with internal developmental and physiological cues. Plant hormones play essential roles in coordinating external and internal signals to elicit the appropriate growth and developmental responses, often in concert, as a means precisely to regulate responses both temporally and spatially. One of the simplest plant hormones is the gaseous molecule ethylene. Ethylene
is best known for its role in the regulation of fruit ripening. Indeed, ethylene has been used for thousands of years by farmers, often unwittingly, to induce ripening.1 It was not until the 20th century, however, that ethylene was recognised as a phytohormone when it was shown to be a plant-produced molecule that induced various physiological effects.2–4 Ethylene is now known to regulate many developmental processes throughout the plant’s life cycle, from germination to senescence, and is also involved in responses to environmental stimuli such as stress and pathogen attack. Many biotechnological applications target ethylene’s role in promoting senescence, fruit ripening and abscission. Ethylene also controls aspects of flower development, the release of dormancy and various defence responses.4–6 At the cellular level, ethylene controls cell elongation, thereby regulating root growth, stem (or hypocotyl) growth and apical hook
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Keywords: ethylene, Arabidopsis, protein complex, transcriptional regulation, microarray, posttranscriptional regulation, ubiquitin, E3-ligase
Dissecting the ethylene pathway of Arabidopsis
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Ethylene signal transduction makes use of positive and negative regulators
formation in seedlings — the latter serving to protect the apical meristem with its stem cells as the seedling pushes up through the soil. These characteristic responses to ethylene formed the basis for a genetic screen used to identify
MAPKKK Cytosol
Nramp metal transporter
Unknown membrane
E3 ligase EIN3/EIL transcription factors
Nucleus
ERF transcription factors
Ethylene responses
Figure 1: Ethylene signal transduction. In the absence of ethylene, constitutive triple response 1 (CTR1) is maintained in an active state by the receptors, which serves to inhibit downstream components and thus the ethylene response. In addition, the transcription factor ethylene insensitive 3 (EIN3) is constantly degraded through the action of EIN3 binding F-box (EBF1) and EBF2 via the proteasome-mediated degradation pathway. Upon binding of ethylene, the receptor inactivates CTR1. This relieves the repression on downstream signalling components, thus allowing for activation of the EIN3/EIL transcription factors and ethylene responses. Ethylene promotes accumulation of EIN3 by repressing the action of EBF1 and EBF2. The subcellular location of components is shown, where known. Ovals represent the active conformations of proteins; rectangles represent inactive conformations of proteins. Cu, copper; ER, endoplasmic reticulum; ERF, ethylene-responsive element binding factor; ETR, ethylene resistant; ERS, ethylene response sensor; MAPKKK, mitogen-activated protein kinase kinase kinase.
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Ethylene receptors
components of the ethylene signalling pathway in the genetic model plant Arabidopsis thaliana. The ethylene signal transduction pathway deduced from genetic analyses can be modelled as a linear pathway from perception to transcriptional regulation (Figure 1). A significant feature of the ethylene signalling pathway is that it incorporates both positive and negative regulators. Ethylene is perceived by a family of receptors that have similarity to histidine kinases, an evolutionarily ancient class of signalling proteins originally identified in bacteria. The ethylene receptors interact with and apparently regulate the activity of constitutive triple response 1 (CTR1), a Ser/Thr kinase that is a negative regulator of ethylene signalling and which actively suppresses the ethylene responses in the absence of ethylene.7 CTR1 has sequence similarity to Raf, a mitogen-activated protein kinase kinase kinase (MAPKKK);7 consequently, a MAP kinase cascade has been postulated to act downstream of CTR1, although the components of this cascade have been the subject of recent debate.8–10 When ethylene binds to the receptors, CTR1 is thought to be inactivated. This relieves the suppression on downstream signalling elements, resulting in the activation of ethylene insensitive 2 (EIN2), a membrane-bound protein with similarity to Nramp metal-ion transporters,11 and the activation of downstream transcriptional regulators.12,13 The most significant family of transcription factors is composed of EIN3 and the EIN3-like (EIL) proteins, of which EIN3 appears to play the predominant role in regulating the ethylene response.13–15 The EIN3/EIL family function in a transcriptional cascade that regulates expression of a variety of genes, including ethylene-responsive factor 1 (ERF1),12,15 one of the ethyleneresponsive element binding factor (ERF) family of transcription factors.16 Kinetic analysis indicates that ethylene’s effect on seedling growth is exerted in two phases. The first phase is a rapid but transient response that occurs within 15
Dissecting the ethylene pathway of Arabidopsis
The response of seedlings to ethylene is biphasic
THE EFFECT OF ETHYLENE ON TRANSCRIPTION Transcriptional profiling has been used by several groups to uncover ethyleneregulated genes. One of the first groups to look at ethylene regulation used a multipronged approach to determine the genes involved in the defence response of Arabidopsis.19 Although only a small proportion of the genome was analysed on these microarrays (2,375 expressed sequence tags [ESTs]), the analysis identified genes that were coordinately regulated by multiple defence elicitors such as salicylic acid, methyl jasmonate
and ethylene.19 This result highlights the cross-talk that occurs between signalling molecules to precisely regulate the defence response. More recently, in an experiment using a 22,000-gene Affymetrix DNA microarray, the effects of ethylene on transcription were analysed in dark-grown seedlings that had received constant exposure to ethylene.20 Approximately 2.9 per cent of the genes tested showed a significant (greater than twofold) change in transcription, the majority of which were repressed.20 Genes involved in many different biological processes, from metabolism to signal transduction, were regulated by ethylene. Several genes containing AP2 domains, including ERF1, were identified as being ethylene induced, four of which contained known plant-specific DNA-binding domains.20 Insertional mutants for each of these four genes were identified and, although single knockout mutants did not demonstrate an altered ethylene response, double mutants were partially ethylene insensitive, thus confirming a role for these genes in mediating the ethylene response.20 In an EST-based microarray study representing 5,955 genes, ethylene’s effect on expression in the leaves of older plants was examined and was also compared with the expression profiles of several known mutants in the ethylene signalling pathway.21 The dominant mutation etr1-1 renders ethylene- resistant 1 (ETR1) incapable of binding ethylene, and results in a plant that is ethylene insensitive.22 By contrast, the loss-of-function mutation ctr1-1 results in a constitutive ethylene response.14 This study focused on genes whose expression is affected by long-term ethylene treatment by examining the aerial parts of adult (24-day-old) plants after treatment with ethylene for 24 hours. The changes in gene expression between the two mutants were examined and compared with wild-type plants treated with ethylene. A higher percentage of ethylene-regulated genes were identified (7 per cent) in this study than in that of Alonso et al.,20 but this may reflect the
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Ethylene transcriptionally regulates genes involved in a wide range of biological processes
minutes of ethylene exposure.17 The first phase is very sensitive to low ethylene levels but is not dose dependent, nor does it require the function of the transcription factors EIN3 and EIL1.18 This suggests that the initial phase may be posttranscriptionally regulated to allow for a rapid response to small changes in ethylene concentration, although further research is necessary to confirm this hypothesis. The first phase is followed by a sustained and slower response that is both dose dependent and EIN3/EIL1 dependent, consistent with a transcriptional requirement for this phase of the growth response.18 The full scope of transcriptional and post-transcriptional regulatory processes on ethylene signalling is only now being appreciated, in part by taking advantage of mutants in the ethylene signal transduction pathway. These mutants have been of great importance in dissecting ethylene’s role in transcriptional regulation via microarray experiments, and also in revealing the role of posttranscriptional regulation of the ethylene response. Proteomic analyses using these mutants have revealed that a receptor complex is responsible for perceiving and transducing the ethylene signal. Recent developments in these topics will be discussed in the following sections, to highlight the role that functional genomics and proteomics is playing in exploring the ethylene signalling pathway.
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There are differing transcriptional responses to short or long term ethylene treatment
that were either repressed or induced by ethylene, but there were several smaller clusters with genes that were distinctly regulated by ethylene after short- or longterm exposure. These experiments confirm that ethylene-regulated transcriptional activation is dose dependent and that short-term ethylene treatments can result in significant transcriptional changes.23 As in the other microarray experiments, genes were identified that are involved in many different biological processes. Interestingly, the cluster of repressed genes primarily encoded proteins involved in metabolism, defence and transport. This study also revealed that several genes involved in ubiquitin/26S proteasome-mediated degradation are ethylene regulated, especially during the early responses to ethylene.23 These experiments have revealed details on the global transcriptional changes that take place in response to ethylene, lending insight into the timedependent nature of the transcriptional response to ethylene and into how mutations in signalling pathway components affect this response. The differences in the number of ethyleneregulated genes uncovered by each of these microarray analyses probably reflect the diverse experimental approaches taken by the researchers. The insights into the global expression patterns of ethylene regulation will be the foundation for future investigations to ascertain ethylene’s roles in the many biological processes under its control.
POST-TRANSCRIPTIONAL REGULATION Precise control of the ethylene response is achieved through complex regulation of the ethylene biosynthesis and signalling pathways. Ethylene has been shown to regulate the transcription of components, such as certain biosynthesis and receptor genes, involved in signalling; however, recent studies have shown that posttranscriptional regulation also plays an important role. One of the post-
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Expression of several genes involved in proteasome-mediated degradation are ethylene regulated
smaller number of genes represented on the array and/or the differences in ethylene treatment conditions. As was found in the study by Alonso et al.,20 ethylene-regulated genes were implicated in many different biological processes, including ethylene biosynthesis and perception, other hormone responses, stress responses and metabolism.21 Surprisingly, comparison of genes regulated in the constitutive ethylene response mutant ctr1-1 with those in wildtype plants treated with ethylene for 24 hours revealed a significant number of genes that were either differentially or oppositely regulated in these sample types.21 These data are indicative of the dose-dependent nature of the ethylene response and also suggest that a negative feedback loop may be in effect.21 Using a similar mutant versus wild-type approach, another group analysed an EST-based microarray representing 6,008 genes in parallel with the polymerase chain reaction-based approach of cDNAamplified fragment length polymorphism (cDNA-AFLP).23 In this case, a different ethylene-insensitive mutant (ein2-1) was used along with the constitutive ethylene response mutant ctr1-1. Loss-of-function mutations in EIN2 result in the strongest ethylene insensitivity of all the ethylene insensitive mutations identified in Arabidopsis,14 and so comparing this genotype to the constitutive ethylene response mutant ctr1-1 is likely to yield the greatest difference in expression of ethylene-regulated genes.23 In this study, the transcriptional changes in adult (19day-old) plants was examined after short(ten-minute) and medium- (6-hour) term ethylene exposure. To avoid complications in gene expression due to circadian rhythms, the authors also discarded genes that were differentially regulated over time in the hope of obtaining a more accurate representation of ethylene-regulated genes.23 A total of 214 genes (3.6 per cent) were found to be ethylene regulated and those genes were placed into distinct clusters of expression patterns.23 The largest clusters were those
Dissecting the ethylene pathway of Arabidopsis
Turnover of the transcription factor EIN3 is regulated by two E3 ligases
downstream component in ethylene biosynthesis, ACC oxidase (ACO), also appears to be regulated by proteasomemediated degradation. Levels of two components involved in activating the E3 ligase, related to ubiquitin (RUB) and RUB-conjugating enzyme (RCE), appear to affect ACO levels and thus activity.34,35 Considering that there are multiple isoforms of both ACS and ACO, further analysis should reveal whether all members of this family are regulated by proteasome-mediated degradation or whether this regulation is specific for certain isoforms. There are several points of posttranscriptional regulation that directly affect ethylene signal transduction. Some mutations in the ethylene-binding domain of ETR1 result in increased protein levels not reflected in changes in mRNA levels, suggesting that at least one of the ethylene receptors is post-transcriptionally regulated.36 Perturbation of ethylene binding with silver causes a similar effect, thus suggesting that ligand binding may regulate receptor turnover.36 This type of ligand-mediated receptor turnover is common in animal systems,37,38 but further analysis of ethylene receptor turnover is required to confirm this proposed mechanism in plants. The activity of a key transcription factor responsible for the ethylene response, EIN3, is also controlled by proteasome-mediated degradation. In the absence of ethylene, EIN3 is continuously degraded, thus preventing activation of its transcriptional targets (Figure 1).39–41 EIN3 protein levels increase in the presence of ethylene, thus enabling this transcription factor to activate its targets and initiate the ethylene response.39–42 Turnover of EIN3 is regulated by two E3 ligases, EIN3 binding F-box (EBF) 1 and EBF2, which mediate ubiquitination of EIN3 and thus promote its degradation. Mutant Arabidopsis plants that are deficient in either or both of these E3 ligases show increased EIN3 levels.39–41 Although EBF1 and EBF2 function in concert to regulate EIN3 levels,
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Proteasome-mediated degradation regulates ethylene biosynthesis
transcriptional mechanisms used in the regulation of ethylene signalling involves ubiquitin/26S proteasome-mediated degradation, a process that has been shown to regulate various physiological processes, including growth and development, hormone responses and defence responses.24,25 Three enzyme complexes, the ubiquitin-activating (E1) enzyme, the ubiquitin-conjugating (E2) enzyme and the ubiquitin-ligating enzyme (E3 ligase), work together to covalently attach ubiquitin moieties to target proteins destined for degradation. E3 ligase confers specificity to the reaction by recognition of degradation targets and by mediating interaction with the E2 enzyme to attach multiple ubiquitin moieties. The polyubiquitinated protein is then recognised and degraded by the 26S proteasome. Within Arabidopsis, there are more than 1,000 potential E3 ligases, each with distinct target specificities, indicating that the plant has a substantial capacity for posttranscriptional regulation through proteasome-mediated degradation.26 Proteasome-mediated degradation is thought to regulate ethylene biosynthesis through one of the key enzymes involved in ethylene biosynthesis 1aminocyclopropane1-carboxylic acid (ACC) synthase (ACS). At least one ACS isoform, ACS4, is targeted to the proteasome-mediated degradation pathway by an N-terminal signal.27 Protein levels of another of ACS isoform, ACS5, are negatively regulated through interaction with ETO1, an E3-ligase component.28–30 Phosphorylation may be involved in ACS turnover,31,32 as has been seen for proteasome-mediated degradation in animal systems.33 Recent data suggest that another ACS isoform, ACS6, is phosphorylated by a MAP kinase (MPK6), and that this phosphorylation results in ACS6 accumulation and a concomitant increase in ethylene production.9 In this case, phosphorylation protects ACS6 from degradation, potentially through a proteasome-mediated pathway.9 The next
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The ethylene receptor ETR1 is localised to the endoplasmic reticulum
THE ETHYLENE RECEPTOR COMPLEX Although hormone receptors are typically localised to the plasma membrane, the ethylene receptor ETR1 is localised to the endoplasmic reticulum (ER) membrane.44 This does not pose a problem for ethylene perception because ethylene can readily diffuse through both aqueous and lipid environments.4 There are several potential advantages for a receptor localised to the ER which may have relevance to ethylene signal transduction. For example, the ER is the site of such cellular functions as calcium storage, protein synthesis, lipid metabolism and defence responses.45–47 The ER is also connected to other organelles through its endomembrane network,47 and could thus provide the receptors with ready access to processes throughout the cell, potentially facilitating communication between organelles and other signalling pathways. CTR1 has also been demonstrated to localise to the ER membrane, even though it is not predicted to contain transmembrane domains.48 This membrane association is apparently due to a physical interaction between CTR1 and the ethylene receptors based on in vivo and in vitro experiments.48–50 It has also been found that ethylene treatment increases the amount of CTR1 protein associated with the ER membrane in a post-transcriptional manner, possibly through stabilisation of the protein by association with the receptors.48 The association between CTR1 and the receptors is required for proper functioning of CTR1 based on mutational analysis. The ctr1-8 mutation was originally isolated in a screen for plants showing a constitutive ethylene response phenotype, indicating that CTR1 was not functional in these plants.51 The ctr1-8 mutation results in a single amino acid change in the Nterminal half of CTR1, in a region distinct from the C-terminal kinase domain. Based on two-hybrid analysis, this mutation abolishes the ability of
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The downstream signalling factor CTR1 physically interacts with the ethylene receptors
differences in their regulation suggest subtly different roles. EBF1 is not transcriptionally regulated by ethylene; however, its basal levels in the plant are higher than those of EBF2,20 suggesting that EBF1 is involved in the initial ethylene response. EBF1 may regulate EIN3 levels in a dose-dependent manner, stimulating turnover at low ethylene concentrations until a certain ethylene threshold has been reached.41 By contrast, EBF2 transcription is induced by ethylene,30,39–41 thus EBF2 may induce degradation of EIN3 at elevated ethylene concentrations. This would facilitate a rapid response to changes in ethylene levels because, upon removal of ethylene, EIN3 would be degraded by EBF2 and thus no longer induce the ethylene response. As previously described, the kinetics of the ethylene response is biphasic, available evidence suggesting that the first phase is post-transcriptionally regulated and responsive to low levels of ethylene.17,18 These characteristics are consistent with EBF1-mediated regulation of EIN3 protein levels, thereby raising the possibility that this initial phase in the ethylene response is regulated by EBF1. The specific mechanism behind ethylene-mediated turnover of EIN3 through EBF1 and EBF2 has yet to be resolved, although several models have been proposed.39–41,43 These models differ in the proposed targets of ethylene’s regulatory action. In one model, ethylene is proposed to affect stability, activity or localisation of EBF1 and EBF2, thus resulting in effects on EIN3 accumulation. Alternatively, ethylene could induce a posttranscriptional modification of EIN3, such as phosphorylation, which may affect the ability of EIN3 to interact with EBF1 or EBF2 and thus enhance the stabilisation of EIN3. Whichever the case, these studies illuminate the complexity of EIN3 regulation and support the theory that this protein is a major control point for the ethylene signalling pathway.
Dissecting the ethylene pathway of Arabidopsis
LOOKING AHEAD The isolation of Arabidopsis mutants with altered ethylene responses was pivotal in the identification of components comprising the ethylene signal transduction pathway. These mutants have played important roles in the analysis and testing of models for ethylene signalling. In this paper, the application of these mutants to microarray analysis and the characterisation of the ethylene receptor/CTR1 complex have been
made apparent. Analysis of how these mutants are affected at the genomic and proteomic levels has barely been initiated and, in the future, will lead to new insights into the mechanism of ethylene signalling and responses. The microarray data gathered to date have been based on the analysis of whole plants or aerial tissues, an approach that averages out changes in gene expression across multiple tissues and, as a result, masks subtle and/or localised effects. Given the range of processes in which ethylene plays a role, it is clear that more focused approaches are required. Greater focus will resolve changes in gene expression for individual tissues and in specific cell types, and will thus result in a clearer picture of the diverse roles that ethylene plays in plant growth and development. Such focused expression pattern profiling has been performed to good effect in the analysis of differing gene expression in specific root cell types56 and points to the fascinating direction that future investigations of this type will take. What is also becoming apparent, even from the limited analyses performed to date, is the importance of posttranscriptional regulation in the modulation of the ethylene response. Many of the proteomics strategies have only begun to be applied to the study of ethylene signalling. For example, an investigation of ethylene-regulated proteins by two-dimensional gel electrophoresis revealed that two proteins, a glutathione S-transferase and a pyrophosphatase-like protein, are significantly induced by ethylene.57 More complete analyses are likely to reveal a greater abundance of ethylene-regulated proteins and, with coordinate microarray data, will yield an improved understanding of the role of posttranscriptional regulation in ethylene signal transduction. Acknowledgments We thank the National Science Foundation and the US Department of Agriculture (NRICGP) for research support.
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The ethylene response is regulated by the interplay between transcriptional and post-transcriptional mechanisms
CTR1 to interact with the ethylene receptor ETR1.51 In addition, in plants with the ctr1-8 mutation, the mutant CTR1 protein is to be no longer found associated with membranes but instead is found in the soluble fraction, apparently due to its inability to associate with the receptors.48 Thus, proper functioning of CTR1, in addition to requiring its kinase activity, also requires a physical association with the ethylene receptors to form a protein complex.48,51 The mechanism behind the activation of CTR1 by the receptors is not clear. CTR1 is similar to the protein kinase Raf, and thus may have similar characteristics — such as a domain that autoinhibits its kinase activity.52 In this model, interaction with the receptor maintains CTR1 in a kinase-active conformation. Upon ethylene binding, the receptor induces a conformational change in CTR1 which allows the autoinhibitory domain to inhibit kinase activity. Another possibility is that the receptors regulate CTR1 activity through phosphorylation, a common means of regulating enzymatic activity. Two ethylene receptors have histidine kinase activity and the others may have Ser/Thr kinase activity.53–55 Further analysis should help to elucidate the mechanism behind the regulation of CTR1 activity. Whichever mechanism is shown to be correct, current evidence indicates that the ethylene receptors and CTR1 function together as a protein complex, and that formation of this complex is essential to ethylene signal transduction.
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Dissecting the ethylene pathway of Arabidopsis
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