Possible modulation of Arabidopsis ETR1 N-terminal signaling by CTR1

The mitogen-activated protein kinase kinase kinase (MAPKKK) Constitutive Triple-Response1 (CTR1) plays a key role in mediating ethylene receptor signaling via its N-terminal interaction with the ethylene receptor C-terminal histidine kinase (HK) domain. Loss-of-function mutations of CTR1 prevent ethylene receptor signaling, and corresponding ctr1 mutants show a constitutive ethylene response phenotype. We recently reported in Plant Physiology that expression of the truncated ethylene receptor Ethylene Response1 (ETR1) isoforms etr11-349 and dominant ethylene-insensitive etr1-11-349, lacking the C-terminal HK and receiver domains, both suppressed the ctr1 mutant phenotype. Therefore, the ETR1 N terminus is capable of receptor signaling independent of CTR1. The constitutive ethylene response phenotype is stronger for ctr1-1 than ctr1-1 lines expressing the etr11-349 transgene, so N-terminal signaling by the full-length but not truncated ETR1 is inhibited by ctr1-1. We address possible modulations of ETR1 N-terminal signaling with docking of CTR1 on the ETR1 HK domain.     

乙烯的生物合成与信号传递

乙烯是气体植物激素, 它在植物的生长发育过程中有很多作用。所以了解乙烯的生物合成及其信号转导是非常重要的。二十年来, 通过筛选有异于正常三重反应的突变体, 人们发现了乙烯信号转导的粗略轮廓。在拟南芥中, 有5个受体蛋白感受乙烯, ETR1、ERS1、ETR2、ERS2、EIN4。它们表现出功能冗余, 是乙烯信号的负调控因子, 在植物体内以二聚体的形式存在。ETR1的N端与乙烯结合时需要 铜离子(Ⅰ)的参与。尽管已经发现ETR1有组氨酸激酶活性, 而其它受体有丝氨酸/苏氨酸激酶活性, 但受体参与乙烯信号转导的机制还不是很清楚。受体与Raf类蛋白激酶CTR1相互作用, CTR1是乙烯反应的负调控因子。CTR1蛋白失活使EIN2蛋白活化。EIN2的N端是跨膜结构域, 与Nramp家族金属离子转运蛋白的跨膜结构域类似。EIN2的C端是一个新的未知结构域, 与乙烯信号途径的下游组分相互作用。EIN3位于EIN2的下游, EIN3和EILs诱导ERF1和其它转录因子的表达, 这些转录因子依次激活乙烯反应目的基因的表达, 表现出乙烯的反应。EIN3受到蛋白酶体介导的蛋白降解途径的调节。由于乙烯是一种多功能的植物激素, 其信号途径与其它信号途径有多重的交叉。     

A MAPK pathway mediates ethylene signaling in plants

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.     

Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr1^1-349 or the dominant ethylene-insensitive etr1-1^1-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr1^1-349 and etr1-1^1-349, with etr1-1^1-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr1^1-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr1^1-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR1^7-560 prevented ethylene receptor signaling, and the CTR1^7-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr1^1-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.The gaseous plant hormone ethylene is perceived by a small family of ethylene receptors. Arabidopsis (Arabidopsis thaliana) has five ethylene receptors that are structurally similar to prokaryotic two-component histidine kinase (HK) proteins. Mutants defective in multiple ethylene receptor genes show a constitutive ethylene response phenotype, which indicates a negative regulation of ethylene responses by the receptor genes (Hua and Meyerowitz, 1998).The receptor N terminus has three or four transmembrane domains that bind ethylene. The GAF (for cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which follows the transmembrane helices, mediates noncovalent receptor heterodimerization and may have a role in receptor cooperation (Gamble et al., 2002; O’Malley et al., 2005; Xie et al., 2006; Gao et al., 2008). The subfamily I receptors Ethylene Response1 (ETR1) and Ethylene Response Sensor1 (ERS1) have a conserved HK domain following the GAF domain. For subfamily II members ETR2, Ethylene Insensitive4 (EIN4), and ERS2, the HK domain is less conserved, and they lack most signature motifs essential for HK activity (Chang et al., 1993; Gamble et al., 1998; Hua et al., 1998; Qu and Schaller, 2004; Xie et al., 2006). Among the five receptors, ETR1, ETR2, and EIN4 have a receiver domain following the HK domain. The ETR1 HK domain may have a role in mediating the receptor signal to downstream components, and the HK activity facilitates the ethylene signaling (Clark et al., 1998; Huang et al., 2003; Hall et al., 2012). The receiver domain can dimerize and could involve receptor cooperation (Müller-Dieckmann et al., 1999). However, differential receptor cooperation occurs between the receiver domain-lacking ERS1 and the other ethylene receptors, which does not support the hypothesis that the domains involve receptor cooperation (Liu and Wen, 2012).Acting downstream of the ethylene receptors is Constitutive Triple Response1 (CTR1), a MEK kinase (mitogen-activated protein kinase kinase kinase) with Ser/Thr kinase activity, and the kinase domain locates at the C terminus. The CTR1 N terminus does not share sequence similarity to known domains and can physically interact with the ethylene-receptor HK domain (Clark et al., 1998; Huang et al., 2003). ctr1 mutants showing attenuated CTR1 kinase activity or the ETR1-CTR1 association exhibit various degrees of the constitutive ethylene-response phenotype. For example, the ctr1-1 and ctr1^btk mutations result from the D694E and E626K substitutions, respectively, in the CTR1 kinase domain, and ctr1-1 shows a stronger ethylene-response phenotype than ctr1^btk, with ctr1-1 having much weaker kinase activity than ctr1^btk (Kieber et al., 1993; Huang et al., 2003; Ikeda et al., 2009). The ctr1-8 mutation results in the G354E substitution that prevents the ETR1-CTR1 association, and the mutant exhibits a constitutive ethylene-response phenotype. Overexpression of the CTR1 N terminus CTR1^7-560, which is responsible for interaction with ethylene receptors, leads to constitutive ethylene responses, possibly by titrating out available ethylene receptors (Kieber et al., 1993; Huang et al., 2003). These studies suggest that CTR1 kinase activity and the interaction of CTR1 with the receptor HK domain may be important to the ethylene receptor signal output in suppressing constitutive ethylene responses.Although the ETR1-CTR1 interaction via the HK domain is essential to the ethylene receptor signal output, evidence suggests that the ETR1 receptor signal output can also be independent of the HK activity or domain. The etr1 ers1 loss-of-function mutant displays extreme growth defects. The etr1[HGG] mutation inactivates ETR1 HK activity, and expression of the getr1[HGG] transgene rescues the etr1 ers1 growth defects, which indicates a lack of association of ETR1 receptor signaling and its kinase activity (Wang et al., 2003). The dominant etr1-1 mutation results in the C65Y substitution and confers ethylene insensitivity (Chang et al., 1993), and the expression of the HK domain-lacking etr1^1-349 and ethylene-insensitive etr1-1^1-349 isoforms partially suppresses the growth defects of etr1 ers1-2. Loss-of-function mutations of subfamily II members do not affect etr1-1^1-349 functions. Therefore, etr1-1^1-349 predominantly cooperates with subfamily I receptors to mediate the ethylene receptor signal output (Xie et al., 2006). Biochemical and transformation studies showing that ethylene receptors can form heterodimers and that each receptor is a component of high-molecular-mass complexes explain how ethylene receptors may act cooperatively (Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010).Reversion To Ethylene Sensitivity1 (RTE1), a Golgi/endoplasmic reticulum protein, was isolated from a suppressor screen of the dominant ethylene-insensitive etr1-2 mutation. The cross-species complementation of the rte1-2 loss-of-function mutation by the rice (Oryza sativa) RTE Homolog1 (OsRTH1) suggests a conserved mechanism that modulates the ethylene receptor signaling across higher plant species (Zhang et al., 2012). RTE1 and OsRTH1 overexpression led to ethylene insensitivity in wild-type Arabidopsis but not the etr1-7 loss-of-function mutant, and expression of etr1^1-349 restored ethylene insensitivity with RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007; Zhang et al., 2010). Coimmunoprecipitation of epitope-tagged ETR1 and RTE1 and Trp fluorescence spectroscopy revealed the physical interaction of RTE1 and ETR1 (Zhou et al., 2007; Dong et al., 2008, 2010). Therefore, RTE1 may directly promote ETR1 receptor signal output through the ETR1 N terminus, but whether RTE1 has an essential role in ETR1 N-terminal signaling remains to be addressed.Currently, the biochemical nature of the ethylene receptor signal is unknown, and the underlying mechanisms of mediation of the ethylene receptor signal output remain uninvestigated. Genetic and biochemical studies suggest that activation of CTR1 by ethylene receptors may suppress constitutive ethylene responses; upon ethylene binding, the receptors are converted to an inactive state and fail to activate CTR1, and the suppression of ethylene responses by CTR1 is alleviated (Hua and Meyerowitz, 1998; Klee, 2004; Wang et al., 2006; Hall et al., 2007). However, this model does not address how the ETR1 N terminus, which does not have the CTR1-interacting site, mediates the receptor signal to suppress constitutive ethylene responses. The receptor signal of the truncated etr1 isoforms may be mediated by other full-length ethylene receptors and then activate CTR1; alternatively, the ETR1 N-terminal signal may be mediated by a pathway independent of CTR1 (Gamble et al., 2002; Qu and Schaller, 2004; Xie et al., 2006). Results showing that mutants defective in multiple ethylene receptor genes exhibit a more severe ethylene-response phenotype than ctr1 and that ctr1 mutants are responsive to ethylene support the presence of a CTR1-independent pathway (Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Huang et al., 2003; Liu et al., 2010).In this study, we investigated whether mediation of ETR1 N-terminal signaling is independent of CTR1 and whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling. The ETR1 N-terminal signaling was not mediated via other full-length ethylene receptors, but the signal of full-length ethylene receptors could be mediated by the ETR1 N terminus independent of CTR1. The ETR1 C terminus may inhibit ETR1 N-terminal signaling, whereby deletion of the C terminus facilitates N-terminal signaling. We propose a model for the possible modulation of ETR1 receptor signaling.     

Phosphatidic acid binds to and inhibits the activity of Arabidopsis CTR1

Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.     

The ethylene signal transduction pathway in Arabidopsis

The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.     

Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1

In Arabidopsis, ethylene is perceived by a receptor family consisting of five members, one of these being ETR1. The N-terminal half of ETR1 functions as a signal input domain. The C-terminal region of ETR1, consisting of a His kinase domain and a putative receiver domain, is likely to function in signal output. The role of the proposed signal output region in ethylene signaling was examined in planta. For this purpose, the ability of mutant versions of ETR1 to rescue the constitutive ethylene-response phenotype of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant line was examined. A truncated version of ETR1 that lacks both the His kinase domain and the receiver domain failed to rescue the triple mutant phenotype. A truncated ETR1 receptor that lacks only the receiver domain restored normal growth to the triple mutant in air, but the transgenic seedlings displayed hypersensitivity to low doses of ethylene. A mutation that eliminated His kinase activity had a modest effect upon the ability of the receptor to repress ethylene responses in air. These results demonstrate that the His kinase domain plays a role in the repression of ethylene responses. The potential roles of the receiver domain and His kinase activity in ethylene signaling are discussed.     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

植物乙烯信号转导研究进展

过去10年,对模式植物拟南芥的分子遗传学研究建立了植物乙烯信号转导线性模型.乙烯结合到受体上,经一条MAPK级联反应和转录级联途径将信号转导而产生乙烯反应.拟南芥乙烯受体家族由5个成员构成,ETR1、ERS1、ETR2、ERS2和EIN4.乙烯受体包括三个结构域:乙烯结合结构域、组氨酸激酶结构域和反应调控结构域.乙烯受体定位于内质网,与CTR1协同负调控乙烯反应.ENI2、EIN3/EIL、ERF1依次位于CTR1下游,正调控乙烯反应.EIN3属于转录激活因子调控蛋白家族,受转录后调控.乙烯稳定EIN3结构,EBF1/EBF2促进EIN3分解.ERF1是转录调控因子家族成员之一,是EIN3/EIL的直接作用目标.     

Loss-of-function mutations in the ethylene receptor ETR1 cause enhanced sensitivity and exaggerated response to ethylene in Arabidopsis

Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response.     

Mutational analysis of the ethylene receptor ETR1. Role of the histidine kinase domain in dominant ethylene insensitivity

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The N-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The C-terminal half of the polypeptide contains domains with homology to histidine (His) kinases and response regulators, signaling motifs originally identified in bacteria. The role of the His kinase domain in ethylene signaling was examined in planta. For this purpose, site-directed mutations were introduced into the full-length wild-type ETR1 gene and into etr1-1, a mutant allele that confers dominant ethylene insensitivity on plants. The mutant forms of the receptor were expressed in Arabidopsis and the transgenic plants characterized for their ethylene responses. A mutation that eliminated His kinase activity did not affect the ability of etr1-1 to confer ethylene insensitivity. A truncated version of etr1-1 that lacks the His kinase domain also conferred ethylene insensitivity. Possible mechanisms by which a truncated version of etr1-1 could exert dominance are discussed.     

The ethylene signal transduction pathway in Arabidopsis

The gaseous hormone ethylene is an important regulator of plantgrowth and development. Using a simple response of etiolatedseedlings to ethylene as a genetic screen, genes involved inethylene signal transduction have been identified in Arabidopsis.Analysis of two of these genes that have been cloned revealsthat ethylene signalling involves a combination of a protein(ETR1) with similarity to bacterial histidine kinases and aprotein (CTR1) with similarity to Raf-1, a protein kinase involvedin multiple signalling cascades in eukaryotic cells. Severallines of investigation provide compelling evidence that ETR1encodes an ethylene receptor. For the first time there is aglimpse of the molecular circuitry underlying the signal transductionpathway for a plant hormone. Key words: Ethylene, plant growth, plant development, regulation, signal transduction, Arabidopsis     

Evidence that CTR1-Mediated Ethylene Signal Transduction in Tomato is Encoded by a Multigene Family Whose Members Display Distinct Regulatory Features

Ethylene governs a range of developmental and response processes in plants. In Arabidopsis thaliana, the Raf-like kinase CTR1 acts as a key negative regulator of ethylene responses. While only one gene with CTR1 function apparently exists in Arabidopsis, we have isolated a family of CTR1- like genes in tomato ( Lycopersicon esculentum ). Based on amino acid alignments and phylogenetic analysis, these tomato CTR1- like genes are more similar to Arabidopsis CTR1 than any other sequences in the Arabidopsis genome. Structural analysis reveals considerable conservation in the size and position of the exons between Arabidopsis and tomato CTR1 genomic sequences. Complementation of the Arabidopsis ctr1-8 mutant with each of the tomato CTR genes indicates that they are all capable of functioning as negative regulators of the ethylene pathway. We previously reported that LeCTR1 expression is up-regulated in response to ethylene. Here, quantitative real-time PCR was carried out to detail expression for LeCTR1 and the additional CTR1 -like genes of tomato. Our results indicate that the tomato CTR1 gene family is differentially regulated at the mRNA level by ethylene and during stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1 -like genes in other species besides tomato was examined through mining of EST and genomic sequence databases.     

Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit,RCN1

Ethylene signaling in Arabidopsis begins with a family of five ethylene receptors that regulate the activity of the Raf-like kinase, CTR1. Recent work to identify novel factors required for modulating ethylene signaling resulted in the isolation of enhanced ethylene response 1 (eer1), a mutant that displays both increased sensitivity and increased amplitude of response to ethylene. Molecular cloning of eer1 reveals that its mutant phenotype results from a loss-of-function mutation in the previously characterized RCN1, one of three PP2A A regulatory subunits in Arabidopsis. Our analysis shows that neither RCN1 expression nor PP2A activity is regulated by ethylene. Instead, we found that Arabidopsis PP2A-1C, a PP2A catalytic subunit previously characterized as interacting with RCN1, associates strongly with the kinase domain of CTR1 in vitro. This likely represents a role for PP2A in modulation of CTR1 activity because an in vitro kinase assay did not reveal phosphorylation of either RCN1 or PP2A-1C by CTR1, indicating that neither of them is a substrate for CTR1. PP2A activity is required for Ras-dependent activation of mammalian Raf, with reductions in PP2A activity significantly compromising the effectiveness of this mechanism. Our genetic and biochemical results suggest that a similar requirement for PP2A activity exists for ethylene signaling, with loss-of-function mutations affecting PP2A activity possibly reducing the effectiveness of CTR1 activation, thus lowering the threshold required for manifestation of ethylene response.     

PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D     

LeCTR2, a CTR1-like protein kinase from tomato, plays a role in ethylene signalling, development and defence

Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein–protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro . Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea , which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I.     

Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), ethylene is perceived by a receptor family consisting of five members. Subfamily 1 members ETHYLENE RESPONSE1 (ETR1) and ETHYLENE RESPONSE SENSOR1 (ERS1) have histidine kinase activity, unlike the subfamily 2 members ETR2, ERS2, and ETHYLENE INSENSITIVE4 (EIN4), which lack amino acid residues critical for this enzymatic activity. To resolve the role of histidine kinase activity in signaling by the receptors, we transformed an etr1-9;ers1-3 double mutant with wild-type and kinase-inactive versions of the receptor ETR1. Both wild-type and kinase-inactive ETR1 rescue the constitutive ethylene-response phenotype of etr1-9;ers1-3, restoring normal growth to the mutant in air. However, the lines carrying kinase-inactive ETR1 exhibit reduced sensitivity to ethylene based on several growth response assays. Microarray and real-time polymerase chain reaction analyses of gene expression support a role for histidine kinase activity in eliciting the ethylene response. In addition, protein levels of the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), which physically associates with the ethylene receptor ETR1, are less responsive to ethylene in lines containing kinase-inactive ETR1. These data indicate that the histidine kinase activity of ETR1 is not required for but plays a modulating role in the regulation of ethylene responses. Models for how enzymatic and nonenzymatic regulation may facilitate signaling from the ethylene receptors are discussed.     

RTE1 is a Golgi-associated and ETR1-dependent negative regulator of ethylene responses

Arabidopsis (Arabidopsis thaliana) RTE1 encodes a membrane protein and negatively regulates ethylene responses. Genetic and transformation studies suggest that the function of the wild-type RTE1 is primarily dependent on ETR1 and can be independent on the other receptors. Ethylene insensitivity caused by the overexpression of RTE1 is largely masked by the etr1-7 mutation, but not by any other receptor mutations. The wild-type ETR1 N terminus is sufficient to the activation of the RTE1 function and the ectopic expression of etr1(1-349) restored ethylene insensitivity conferred by 35SgRTE1 in etr1-7. The RTE1 N terminus is not essential to the etr1-2 function and the expression of rte1(NDelta49), which has an N-terminal deletion of 49 amino acid residues, restored ethylene insensitivity in etr1-2 rte1-2. The ectopic expression of GREEN FLUORESCENT PROTEIN (GFP)-RTE1 conferred ethylene insensitivity in wild type and the GFP fusion displayed fast movement within the cytoplasm. The GFP-RTE1 and EYFP-NAG proteins colocalized and the Brefeldin A treatment caused aggregation of GFP-RTE1, suggesting RTE1 is a Golgi-associated protein. Our results suggest specificity of the RTE1 function to ETR1 and that endomembranes may play a role in the ethylene signal transduction.