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Annual Plant Reviews (2012) 44, 147–168 http://onlinelibrary.wiley.comdoi: 10.1002/9781118223086.ch6

Chapter 6

ETHYLENE SIGNALLING: THECTR1 PROTEIN KINASESilin Zhong1 and Caren Chang2

1Boyce Thompson Institute for Plant Research, Cornell University, Ithaca,NY 14853, USA2Department of Cell Biology and Molecular Genetics, University of Maryland,College Park, MD 20742, USA

Abstract: CONSTITUTIVE TRIPLE RESPONSE (CTR1) is an important negativeregulator of ethylene signalling that was first identified by the isolation of a con-stitutive ethylene-response mutant in Arabidopsis. In the absence of ethylene treat-ment, ctr1 mutants exhibit the same phenotypes as ethylene-treated wild-typeplants. Arabidopsis has only one CTR1 gene in ethylene signalling, whereas intomato (Solanum lycopersicon), three CTR1 homologues are involved in ethylenesignalling and display differential gene expression patterns that might reflect spe-cific functions. CTR1 encodes a Raf-like serine/threonine (Ser/Thr) protein kinasewith an N-terminal regulatory domain and a C-terminal kinase domain. CTR1 actsdownstream of the ethylene receptors and upstream of EIN2. When the receptorsperceive ethylene, CTR1 kinase activity is shut off, thereby leading to responses.CTR1 has been shown to physically associate with the ethylene receptors at theendoplasmic reticulum membrane, but the biochemical mechanisms of CTR1 reg-ulation remain unclear at this point. The downstream substrate(s) of CTR1 areunknown as well. CTR1 has the highest sequence similarity to Raf protein kinases,so it has been long assumed that CTR1 functions in a mitogen-activated proteinkinase (MAPK) cascade. The existence of a MAPK cascade in ethylene signallinghas been controversial however, and functional similarities between CTR1 andMAP kinase kinase kinase (MAPKKKs) remain speculative. Thus, the remainingcritical questions surrounding CTR1 are the regulation of CTR1 and how the signalis transduced from CTR1 to downstream components. The study of non-ethylene-response phenotypes in ctr1 mutants has been useful for revealing interactionsbetween ethylene signalling and other plant signals, including gibberellin, auxinand abscisic acid.

Annual Plant Reviews Volume 44: The Plant Hormone Ethylene, First Edition. Edited by Michael T. McManus.C© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

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148 � The Plant Hormone Ethylene

Keywords: ethylene; kinase; phosphorylation; signal transduction; mitogen-activated protein kinase; negative regulator

6.1 Introduction

The CONSTITUTIVE TRIPLE RESPONSE (CTR1) locus was uncovered twodecades ago and has been shown to be an important negative regulator ofethylene signal transduction. As presented in this chapter, much is knownabout the CTR1 gene, its homologues and the CTR1 protein. This chapterincludes a historical overview of the molecular cloning of CTR1, the charac-terization of ctr1 mutant phenotypes and gene family members, the analysisof CTR1 biochemical activity and a discussion of the potential regulation ofCTR1 activity and possible downstream effectors.

6.2 Discovery of CTR1, a negative regulator of ethylenesignal transduction

6.2.1 Isolation of the Arabidopsis CTR1 mutant

The ethylene signal transduction pathway began to be dissected at the molec-ular level in the late 1980s based on the isolation of ethylene-response mutantsin the model plant Arabidopsis thaliana (Bleecker et al., 1988; Guzman & Ecker,1990; Roman et al., 1995). The identification of such mutants has largely reliedon a singular phenotype known as the ‘triple response’, which is exhibitedby dark-grown seedlings germinated in the presence of ethylene (Neljubow,1901). The triple response phenotype consists of a shortened and thickenedhypocotyl, an exaggerated apical hook, a shortened root and a proliferationof root hairs (Figure 6.1(a)). Importantly, this phenotype is highly specific toethylene; no other single hormone is known to produce the complete tripleresponse phenotype.

The CTR1 locus was discovered by screening for mutants that exhibit thetriple response phenotype in the absence of ethylene treatment (Figure 6.1(a)).This is in contrast to ethylene-insensitive mutants, such as etr and ein mutants,which lack the triple response when treated with ethylene. Two classes ofconstitutive ethylene-response mutants were found. One caused ethyleneoverproduction and was designated eto for ethylene overproducer. The other didnot overproduce ethylene and could not be rescued by chemical inhibitors ofeither ethylene biosynthesis or ethylene binding, indicating that this mutantclass was involved in ethylene signalling as opposed to biosynthesis. Theselatter mutants fell into a single complementation group and were mappedto a single locus named CTR1 located on Arabidopsis chromosome 5 (Kieberet al., 1993).


Ethylene Signalling: the CTR1 Protein Kinase � 149

(a) (b)

airair

ethylene

ethylene

WT ctr1 ein2WT ctr1 ein2

Figure 6.1 CTR1 is a negative regulator of ethylene responses. (a) The constitutivetriple response of the Arabidopsis ctr1 mutant is shown in comparison to the wild typeand the ein2 ethylene-insensitive mutant. Dark-grown seedlings were germinated withand without ethylene treatment for 4 days. For ethylene treatment, the ethyleneprecursor 1-aminocyclopropane-1-carboxylic acid (ACC) was added to the growthmedium at a concentration of 20 �M. The wild type exhibits the triple response onlywhen treated with ethylene, whereas the ctr1 mutant displays the triple response bothwith and without ethylene treatment. (b) The inhibition of leaf cell expansion in the ctr1rosette is shown in comparison to the wild type and the ein2 ethylene-insensitive mutant.Plants were grown in air versus 1-ppm ethylene. (Panel (b) is reprinted from Kieber et al.,1993.) (For a colour version of this figure, please see Plate 6.1.)

All known ctr1 mutations are recessive to wild type, suggesting that theycause a loss of CTR1 function that confers constitutive activation of ethyleneresponses. Based on this, it was deduced that the wild-type CTR1 gene is anegative regulator of ethylene signal transduction, acting to prevent ethy-lene responses (if CTR1 were a positive regulator, then the loss-of-functionwould have resulted in ethylene insensitivity). It is now known that manysignalling pathways in plants involve negative as well as positive regulation,as recognized a number of years ago by Bowler and Chua (1994).

6.2.2 CTR1 mutant phenotypes in Arabidopsis

ctr1 mutants exhibit constitutive ethylene-response phenotypes throughoutthe life cycle, indicating that CTR1 acts in an ethylene-response pathwaythat is common to most tissues and developmental stages. Besides the con-stitutive triple response in the seedling, ctr1 mutants have a smaller rosette(Figure 6.1(b)) and a more compact inflorescence than the wild type; ctr1plants also exhibit longer petioles, delayed flowering (by 1–2 weeks) and aless extensive root system (Kieber et al., 1993). Many of these phenotypes



Figure 6.2 Genetic pathway showing position of CTR1. CTR1 has a central position inthe ethylene-response pathway as determined by genetic epistasis in Arabidopsis. CTR1acts at or downstream of the ethylene receptors and at or upstream of EIN2.

can be phenocopied by wild-type plants treated with ethylene. Genes thatare normally up-regulated by ethylene, such as CHITINASE, are constitu-tively expressed in ctr1 mutants (Kieber et al., 1993). As discussed later in thischapter, the altered growth phenotypes might be related to crosstalk betweenethylene and the phytohormone gibberellin (GA). Somewhat unexpectedly,the ctr1–1 mutant exhibits insensitivity to ethylene-induced leaf senescence,but this is consistent with the finding that wild-type plants treated continu-ously with ethylene show reduced leaf senescence (Jing et al., 2005). In ctr1flowers, the gynoecium elongates early, often protruding from the flower bud(Kieber et al., 1993). This developmental defect might explain why the earlyflowers of ctr1 plants are often infertile. There is also reduced transmission ofctr1 mutant alleles through the female gametophyte (Kieber & Ecker, 1994).

6.2.3 Placement of CTR1 in the ethylene-response pathway

Placement of CTR1 within the ethylene-signalling pathway was establishedon the basis of double mutant analyses between ctr1 and existing ethylene-insensitive mutants (Kieber et al., 1993; Hua et al., 1995; Roman et al., 1995; Huaet al., 1998; Sakai et al., 1998). Double mutants of ctr1 and each of the dom-inant ethylene-insensitive ethylene receptor mutants exhibit a constitutivetriple response phenotype, indicating that CTR1 acts at or downstream of allfive ethylene receptors (Figure 6.2). Double mutant analyses with the reces-sive ethylene-insensitive mutants ein2 and ein3 indicate that CTR1 acts at orupstream of both EIN2 and EIN3. EIN2 encodes a protein with weak similarityto the NRAMP (natural resistance-associated macrophage proteins) family ofmetal ion transporters, while EIN3 is a nuclear-localized transcription factor,as reviewed by Stepanova and Alonso (2009).

It is worth noting that ctr1–3 and ctr1–9 (likely null mutants) can still re-spond to ethylene as exhibited by enhancement of the constitutive tripleresponse phenotype upon treatment with the hormone (Larsen & Chang,2001; Huang et al., 2003). In addition, the rosette leaves of the ctr1–1 loss-of-function mutant show a senescence response to ethylene (Jing et al., 2005).While these responses could be due to residual wild-type CTR1 functionin these mutants, an alternate possibility is that there is an unidentified al-ternate signalling pathway(s) that bypasses CTR1. According to the modelof ethylene receptor signalling (see also Chapter 5), in the absence of ethy-lene binding, the ethylene receptors negatively regulate ethylene signallingby activating CTR1. When ethylene receptor signalling is turned ‘off’ (upon



ethylene binding), then CTR1 signalling is turned ‘off’. Interestingly, the etr1ers1 double ethylene receptor null mutant (in which signalling by these tworeceptors is ‘off’) displays more severe phenotypes than the ctr1–3 mutant,perhaps reflecting a degree of CTR1-independent signalling by the ETR1 andERS1 ethylene receptors (Qu et al., 2007). Consistent with this, it appearsthat ctr1–3 does not completely suppress the gain-of-function signalling ofthe dominant ethylene-insensitive etr1–1 mutant, i.e. the etr1–1 ctr1–3 mutantdoes not have a constitutive triple response as severe as the ctr1–3 mutantalone (Kendrick & Chang, unpublished).

6.3 CTR1 Encodes a serine/threonine protein kinase

6.3.1 Molecular cloning and sequence analysisof the Arabidopsis CTR1 gene

Arabidopsis CTR1 was the first gene in the ethylene-signalling pathway tobe cloned and was accomplished by a combination of genetic mapping andplasmid rescue of a ctr1 T-DNA insertion (Kieber et al., 1993). The predictedprotein sequence indicates that CTR1 is a protein kinase of 821 residues(∼90 kDa) (Figure 6.3). Its C-terminal portion is a catalytic domain that con-tains all 11 conserved sub-domains of protein kinases. CTR1 has the highestsimilarity (41% sequence identity in the kinase domain) to the Raf family ofSer/Thr protein kinases. Raf kinases regulate mitogen-activated protein ki-nase (MAPK) cascades in mammals. The MAPK cascade, found in many sig-nalling pathways in eukaryotes from yeast to humans, involves a conservedmodule of three protein kinases consisting of a MAP kinase kinase kinase(MAPKKK) (such as Raf-1) that activates a MAP kinase kinase (MAPKK),which in turn activates a MAPK (Ichimura et al., 2002).

The CTR1 N-terminus (∼550 residues) is less conserved with Raf and isthought to comprise a regulatory domain. Sequence similarity to Raf ex-tends only ∼280 residues upstream of the kinase domain and includes aSer-rich region (Kieber et al., 1993). In common with the N-terminus of Raf,the CTR1 N-terminus is rich in glycine and Ser/Thr residues. Unlike Raf, theCTR1 N-terminus lacks a canonical cysteine finger and contains a consensus

821

Kinas domainRegulatory domainN C

CN box1

Figure 6.3 CTR1 protein structure. Arabidopsis CTR1 is an 821-amino acid protein witha conserved protein kinase domain at the C-terminus (Kieber et al., 1993). The N-terminaldomain is a putative regulatory domain comprising ∼550 residues and including a CNbox that may be involved in protein–protein interactions (Huang et al., 2003).



nucleotide triphosphate-binding P-loop motif, which is not usually presentin protein kinases.

Although CTR1 is commonly called a Raf-like kinase, the extent to whichCTR1 and Raf kinases are functionally similar remains unclear. As discussedin later sections of this chapter, the regulation of CTR1 has striking parallelswith Raf, but there are distinct differences as well. Later in the chapter, wealso describe the physical association of the CTR1 N-terminal domain withthe ethylene receptors. This association appears to involve a conserved regionof the N terminus named the CN box, which is invariant among plant CTRs(Huang et al., 2003).

A number of ctr1 mutant alleles have been isolated, including T-DNAinsertions, nonsense mutations, missense mutations and a small deletion thatcauses a frameshift (Kieber et al., 1993; Gibson et al., 2001; Huang et al., 2003;Ikeda et al., 2009). These mutations are all recessive to wild type. Severalmissense mutations result in amino acid substitutions of CTR1 carboxyl-terminal residues that are conserved in protein kinases. One allele calledctr1btk (which was initially known as beatnik) encodes an E626K substitutionin a non-conserved residue of the kinase domain (Ikeda et al., 2009). Within theN-terminal domain, there is only one known missense allele, ctr1–8, whichencodes a G354E substitution of a conserved residue within the CN box(Huang et al., 2003).

6.3.2 CTR1 biochemical activity

CTR1 has been shown to have Ser/Thr protein kinase activity in vitro, and lossof this activity correlates with the constitutive ethylene-response phenotype(Huang et al., 2003). Kinase assays have been carried out using recombinantfull-length CTR1 or just the CTR1 kinase domain alone, expressed in andpurified from insect cells. Both versions exhibited autophosphorylation, witha fraction of autophosphorylation occurring as an inter-molecular reaction.The autophosphorylation appears to occur at multiple sites on CTR1. CTR1is also capable of phosphorylating myelin basic protein (MBP), which isfrequently used as an artificial phosphorylation substrate for Raf-1. AtMEK1(an Arabidopsis MAPKK homologue), however, is phosphorylated only poorlyby CTR1, suggesting that AtMEK1 is not a physiological substrate for CTR1.In all cases, phosphorylation has been detected on Ser and Thr residues; nophosphorylation has been detected on tyrosine residues. The Km value ofCTR1 (9.1 �M) for ATP is similar to that of Raf-1 (11.6 �M). As with Raf-1activity, CTR1 activity is observed when either Mg2+ or Mn2+ is provided asthe required divalent cation.

Huang et al., (2003) also assayed the activity of CTR1–1 and CTR1–8 mutantversions, which carry amino acid substitutions in the C-terminal catalytic andN-terminal regulatory domains, respectively. The ctr1–1 mutant has a strongconstitutive ethylene-response phenotype, while ctr1–8 has a weaker pheno-type. For the ctr1–1 product, the level of in vitro phosphorylation was found



to be <0.1% that of wild-type CTR1, consistent with the ctr1–1 substitution(D694E) disrupting catalytic activity. From this loss of activity, it was de-duced that CTR1 kinase activity was required to repress ethylene responses.In contrast, ctr1–8, encoding the G354E substitution of a conserved residue inthe N-terminal domain, has no detrimental effect on intrinsic kinase activity.Therefore, the ctr1–8 mutation is thought to disrupt the regulation of CTR1kinase activity, inhibiting its activation.

The ctr1btk mutation E626K weakens but does not abolish in vitro kinaseactivity (Ikeda et al., 2009). Notably, ctr1btk has a short root with root hairs athyper-polar positions, but does not display other ctr1 phenotypes, such asthe constitutive triple response.

6.4 The CTR1 gene family

6.4.1 The CTR multi-gene family in tomato

Arabidopsis CTR1 is a B3 sub-group member of the Arabidopsis MAPKKKsuperfamily (Ichimura et al., 2002). Among the six members of the B3 sub-group, there is no indication of functional redundancy, suggesting that CTR1is the only member involved in ethylene signalling in Arabidopsis. In contrast,multiple CTR-like genes have been identified in other plant species, includingmajor crops such as rice, fruit species such as tomato (Leclercq et al., 2002;Adams-Phillips et al., 2004) and ornamental plants such as rose (Muller et al.,2002) (Figure 6.4). The role of ethylene in fruit ripening has been intensivelystudied in tomato, an important model for fleshy fruit development. Tomatohas four CTR-like genes, three of which have been shown to take part in ethy-lene signalling (Leclercq et al., 2002; Adams-Phillips et al., 2004; see Chapter11). LeCTR1 was identified as an ethylene-inducible cDNA by mRNA dif-ferential display (Zegzouti et al., 1999), and LeCTR2, LeCTR3 and LeCTR4were isolated from cDNA libraries using AtCTR1 as a hybridization probe(Adams-Phillips et al., 2004; Lin et al., 2008). In common with their Arabidop-sis orthologs, the LeCTRs possess a carboxyl-terminus that shares sequencehomology with the Raf family Ser/Thr protein kinases. They all contain theprotein kinase ATP-binding site, the Ser/Thr protein kinase active site andthe 11 sub-domains common to all known protein kinases. While their N-terminal regions share less sequence homology with Arabidopsis CTR1 (50%identity), the CN box in the N-terminus is conserved in all tomato CTRs.

6.4.2 Functional roles of tomato CTR genes

Ethylene plays a critical role in determining the timing of fruit ripening. It iswell known that the application of ethylene promotes fruit ripening, whereasdown-regulation of ethylene biosynthesis genes or a mutation blocking



Figure 6.4 The CTR-like multi-gene family. Phylogenetic analysis of plant CTR1-likeproteins. The cucumber (Cs), tomato (Le), rice (Os), poplar (Pt), rose (Rh), Sorghum (Sb)and maize (Zm) CTR-like protein sequences were obtained from Genbank and alignedwith members of the Arabidopsis mitogen-activated protein (MAP) kinase kinase kinase(MAPKKK) B3 sub-groups, including CTR1 and EDR1, using ClustalW2. Raf-1 was used asthe outgroup. The bootstrap tree was generated using MEGA 4.1 package.

ethylene perception delays fruit ripening (Alexander & Grierson, 2002; seeChapter 11). LeCTR1 is the most abundant CTR-like gene expressed duringtomato fruit development. Silencing of LeCTR1 in fruit leads to the early onsetof ripening (Fu et al., 2005), and in silenced seedlings there is a constitutiveethylene-response-like phenotype and up-regulation of ethylene-induciblegenes (Liu et al., 2002). This suggests that in the absence of LeCTR1, the tomatoethylene response is de-repressed, consistent with the role of CTR1 as a



negative regulator of ethylene signalling. LeCTR2 has higher sequencesimilarity with Arabidopsis ENHANCED DISEASE RESPONSE1 (EDR1),which encodes a CTR1-like Ser/Thr protein kinase that negatively regu-lates defence responses, programmed cell death and ethylene-induced leafsenescence (Tang et al., 2005). Tomato plants overexpressing the LeCTR2amino-terminus develop an enhanced hypersensitive response to the fun-gal pathogen Botrytis cinerea (Lin et al., 2008). Ectopic expression of LeCTR2in wild-type Arabidopsis is able to confer an enhanced senescence phenotypesimilar to that of EDR1 overexpression lines.

Despite the remarkable conservation of the ethylene-signalling pathwayand sequence homology of genes between tomato and Arabidopsis, uncer-tainties remain as to whether members of the tomato CTR gene family areredundant or have specific biological functions. For example, LeCTR3 is thegene most similar to AtCTR1. A unique ATP/GTP-binding site in both of theirencoded amino-termini is absent in other tomato CTR family members. Inaddition, ectopic expression of LeCTR3 can fully complement the Arabidopsisctr1 loss-of-function mutant, whereas LeCTR1 and LeCTR4 are only able topartially rescue the ctr1 mutant (Adams-Phillips et al., 2004). This suggestssome functional redundancy among the LeCTRs but might also reflect differ-ences in ethylene-signalling capacities, at least when expressed in Arabidopsis.For instance, the tomato CTRs might not associate equally with the Arabidop-sis receptors due to divergence of their amino-termini. It is also possible thatthese additional CTR genes have evolved to regulate a subset of ethyleneresponses or unique developmental processes that might not be present inArabidopsis.

6.4.3 Transcriptional regulation of CTR-like genes

Tomato CTR-like genes have distinct patterns of expression, in contrast toArabidopsis CTR1, which is constitutively expressed in all tissues that havebeen examined and whose expression is unaffected by ethylene treatment(Kieber et al., 1993; Gao et al., 2003). LeCTR1 is highly expressed in ripeningfruit, while LeCTR3 and LeCTR4 transcripts accumulate to higher levels inleaves than in fruit and remain relatively constant during fruit development(Adams-Phillips et al., 2004). In addition, the expression of LeCTR1 is inducedby ethylene treatment, whereas LeCTR3 and LeCTR4 expression does notrespond to exogenous ethylene (Adams-Phillips et al., 2004). The expressionof LeCTR2 appears to be negatively correlated with ethylene signalling, sinceLeCTR2 transcripts are more abundant in the non-ripening mutants never-ripe (nr) and ripening inhibitor (rin) compared to wild type (Lin et al., 2008)(Nr is an ethylene-insensitive ethylene receptor mutant, and the rin mutantis defective in ethylene production during fruit development; see Chapter 11for further details).

Differential regulation of CTR is not unique to tomato. Various CTR-like gene expression patterns have been reported in many plant species. In



kiwifruit (Actinidia deliciosa) flesh tissue, both AdCTR1 and AdCTR2 have adecreased expression pattern during fruit development, while only AdCTR1increases at the climacteric stage and neither responds to ethylene treatment(Yin et al., 2008). Peach (Prunus persica) is another climacteric fruit that re-quires a burst of ethylene production at an early stage of ripening. It has beensuggested that the peach cultivar ‘Stony Hard’ bearing crispy (non-ripe) fruitcontains a defect in fruit ethylene biosynthesis (Begheldo et al., 2008). Whenexposed to low-temperature storage, the fruit has a sudden increase of ethy-lene production and a transient up-regulation of peach CTR1 expression,followed by eventual fruit softening. It should be noted that not all fruitsregulate CTR at the transcriptional level. For example, the RNA level of CTRin peach, apple and pear is rather constant during early ripening stages (Cinet al., 2006; Wiersma et al., 2007). Ethylene is also known to be a key regulatorof flower opening, which is a vital developmental event for spermatophytes.Ethylene treatment of cut rose (Rosa hybrida) hastens the flower opening pro-cess and induces early petal abscission (Muller et al., 2002; Ma et al., 2006).Both rose genes, RhCTR1 and RhCTR2, are up-regulated by ethylene treat-ment in petals, while the application of the ethylene action inhibitor 1-MCPsubstantially suppresses expression.

Although a large body of experimental data has been gathered regardingthe transcript levels of CTR-like genes, little is known about CTR-like proteinlevels in species other than Arabidopsis. In Arabidopsis, ethylene treatmentappears to have no effect on CTR1 transcript levels, yet CTR1 protein lev-els show a slight increase after ethylene treatment (Gao et al., 2003). In anopposite fashion, transcripts of the tomato ethylene receptor genes NR andLeETR4 increase during ripening, yet the levels of the corresponding proteinsactually decrease (Kevany et al., 2007). There is evidence that ethylene bind-ing can lead to ethylene receptor degradation through a proteasome-basedpathway (Chen et al., 2007; Kevany et al., 2007). Like CTR1, the ethylenereceptors are negative regulators of ethylene signalling. Hence, reducing re-ceptor protein levels could be considered as an enhancement of ethylenesignal output favouring fruit ripening. It is thus tempting to speculate thattomato LeCTR1, with a ripening-associated expression profile, could be sub-jected to a similar post-translational regulation. On the other hand, the slightincrease in Arabidopsis CTR1 protein levels might facilitate a rapid reversalof ethylene signalling through the activation of CTR1 when the levels of thehormone declines. In other species, the existence of a CTR gene family withdistinct regulatory mechanisms raises the possibility that plants may havemodified these mechanisms as an adaptation to more diverse biological rolesof ethylene as appropriate for their growth and survival.

6.5 Regulation of CTR1 activity

The molecular mechanisms by which CTR1 kinase activity is regulated byethylene remain unclear. As mentioned earlier, genetic epistasis analysis has



CTR1

ETR1

ER

C

-

Ethylene

N-

Cu2+

N-

Cu2+

-

C

N-Ethylene

responsesOFF

Ethyleneresponses

ON

KDC

-

PA?

14-3-3?

?

?PP2A subunit?

Cytoplasm

MAPKK?

?

KD

Figure 6.5 Summary of CTR1 regulation in Arabidopsis. CTR1 is known to physicallyassociate with the ethylene receptors in Arabidopsis (Clark et al., 1998; Cancel & Larsen,2002; Gao et al., 2003) and tomato (Zhong et al., 2008). Left: In the absence of ethylenebinding, the ethylene receptor ETR1 (or other ethylene receptors) activates the CTR1kinase domain (KD) by an unknown mechanism, which leads to repression of ethyleneresponses. Protein phosphatase 2A might play a role in activating CTR1 (Larsen & Cancel,2003). The immediate downstream target of CTR1 signalling has not been identified, butcould involve a mitogen-activated protein (MAP) kinase kinase kinase (MAPKK) (Hahn &Harter, 2009). Right: When ethylene is bound, receptor activation of CTR1 kinase activityis terminated by an unknown mechanism. CTR1 inactivation may involve induction of aconformational change and/or auto-inhibition of the KD by the CTR1 N-terminal domain,and may be stabilized by 14–3-3 proteins. Phosphatidic acid (PA) may inhibit CTR1 kinaseactivity and disrupt the interaction with ETR1 (Testerink et al., 2007). Figure adaptedfrom Kendrick & Chang, 2008. (For a colour version of this figure, please see Plate 6.2.)

placed CTR1 at or downstream of the ethylene receptors in the ethylene-signalling pathway. Analyses of mutant phenotypes have indicated that inthe absence of ethylene, CTR1 is activated by the receptors to repress ethy-lene signalling (Figure 6.5). Ethylene binding causes the receptors to turn offCTR1 kinase activity, resulting in de-repression of the pathway (Huang et al.,2003). Thus, a gain-of-function mutant receptor (e.g. ETR1–1), which can-not bind ethylene, constitutively activates CTR1, leading to dominant ethy-lene insensitivity (Bleecker et al., 1988), while plants with multiple receptor



loss-of-function mutations (e.g. the etr1–7 ers1–3 double mutant) are unableto activate CTR1, resulting in a constitutive ethylene-response phenotype(Hua & Meyerowitz, 1998; Qu et al., 2007). The kinase activity of CTR1 isknown to be essential for the biological function of CTR1, since mutations dis-rupting the kinase catalytic domain result in constitutive ethylene response.

6.5.1 Physical association of CTR1 with ethylene receptors

A significant advance in understanding CTR1 function was the discovery inArabidopsis that the N-terminus of the protein can physically interact with theethylene receptors both in vitro and in vivo (Clark et al., 1998; Gao et al., 2003).As described in Chapter 5, the ethylene receptors contain a conserved histi-dine kinase-like domain and, in some cases, a canonical receiver domain at theC-terminus (Chang et al., 1993; Bleecker, 1999). The histidine kinase domain ofArabidopsis ETR1 and ERS1 can associate with CTR1 in the yeast two-hybridassay, and the presence of the receiver domain in ETR1 significantly enhancesthis interaction (Clark et al., 1998). Recently, a similar physical association hasbeen demonstrated for tomato CTRs and tomato ethylene receptors (Lin et al.,2008; Zhong et al., 2008).

In the mammalian Ras–Raf signalling model, the small G-protein Ras isactivated by the receptors upon ligand binding and then interacts with theN-terminus of Raf. This interaction recruits Raf to the plasma membranewhere its C-terminal kinase domain is activated to trigger the MAPK phos-phorylation cascade (Wellbrock et al., 2004). Although plants do not have aRas homologue, the 3D structure of the receiver domain, which is present inthe ETR1 receptor, is quite similar to that of Ras (Chen et al., 1990). Neverthe-less, it has never been known for a Raf-like kinase to be physically coupledwith a histidine kinase-like receptor, as histidine protein kinases are prevalentin prokaryotes but essentially absent in higher eukaryotes.

Interestingly, the interaction of CTR1 with the subfamily II receptor ETR2in the yeast two-hybrid assay is much weaker (Cancel & Larsen, 2002). Com-pared to subfamily I receptors (ETR1 and ERS1), the histidine kinase domainsin subfamily II receptors (ETR2, ERS2 and EIN4) are diverged and lack someessential residues for histidine kinase activity. Only ETR1 autophosphory-lates a histidine residue in vitro; ERS1 can phosphorylate on both histidineand Ser, while the remaining Arabidopsis ethylene receptors phosphorylate Ser(Gamble et al., 1998; Moussatche & Klee, 2004). It is unclear whether receptorkinase activity is required for the ability to associate with CTR1, since ETR1carrying a mutated histidine kinase domain is still able to pull down CTR1in vitro (Gao et al., 2003). However, the subfamily II receptor might indirectlyregulate CTR1 through subfamily I receptors via intra-receptor interaction, asthe receptors can form both homo- and heterodimer and even higher orderclusters at the endoplasmic reticulum (ER) membrane (Gao et al., 2008; Grefenet al., 2008; Chen et al., 2010).



The activation of CTR1 potentially involves phosphorylation by the re-ceptors, given that the receptors are protein kinases. However, the role ofreceptor protein kinase activity in ethylene signalling is unclear (as discussedin Chapter 5). Even if ethylene binding activates or inhibits receptor kinaseactivity, there is no evidence, as yet, that CTR1 is a phosphorylation substratefor the receptors despite their physical interaction. As mentioned in Chapter4, the biochemical mechanism of ethylene receptor signalling has yet to beelucidated. In any case, the shutting off of ethylene receptor signalling couldserve to dislocate CTR1 from the receptors or the membrane and/or alter theconformation of CTR1, consequently inactivating the protein (Figure 6.5).

6.5.2 Membrane localization of CTR1

The localization or active recruitment of CTR1 to the membrane may play animportant role in its activation, as in the Raf model. CTR1 is associated withthe ER fraction where it co-localizes with the ETR1 ethylene receptor (Gaoet al., 2003). This interaction with the ethylene receptors could be responsiblefor CTR1 membrane localization, since CTR1 has no predicted transmem-brane domain (Kieber et al., 1993) and dissociates from the ER membrane inthe multiple receptor null mutant (Gao et al., 2003). Such membrane recruit-ment could be important for regulating CTR1 activity and/or downstreamsignalling. For example, membrane localization could place CTR1 in contactwith regulatory elements other than the ethylene receptors and/or phospho-rylation substrates.

Studies with the ctr1–8 mutant allele suggest that association of the N-terminus of CTR1 with the ethylene receptors is essential not only for mem-brane localization of CTR1, but also for the activation of CTR1 kinase activity(Huang et al., 2003). As mentioned earlier, the ctr1–8 mutation, encoding aG354E substitution in the CN box of the CTR1 N-terminal domain, does notdisrupt protein kinase activity. This mutation, however, was found to dis-rupt the interaction between CTR1 and the ethylene receptor (Huang et al.,2003). Since the ctr1–8 mutant phenotype is comparable to that of other ki-nase inactive ctr1 alleles, the kinase activity in CTR1–8 apparently fails to beswitched on. Overexpression of the N-terminus of CTR1 alone (without thekinase domain) reproduces a CTR1 loss-of-function phenotype. This mightbe caused by failure of the native full-length CTR1 protein to be activated bythe ethylene receptors due to competition with the overexpressed N-terminusfor docking sites on the receptors (Huang et al., 2003).

6.5.3 An inhibitory role for the CTR1 N-terminus?

In the mammalian Raf-signalling model, the N-terminus of Raf inhibits theactivity of its C-terminal kinase domain by an inter-molecular interaction(Wellbrock et al., 2004). This is supported by the observation that its N-terminus can associate in vitro and co-immunoprecipitate in vivo with the



C-terminal kinase domain and that truncated forms of Raf lacking the auto-inhibitory N-terminal domain are constantly active (Chong & Guan, 2003).Despite the lack of sequence homology between CTR1 and Raf in their N-termini, in vitro binding assays show that the Arabidopsis CTR1 N-terminuscan associate with its own kinase domain (Larsen & Cancel, 2003; Shockey &Chang, unpublished data). One report has shown that transient expressionof the CTR1 C-terminal kinase domain without the N-terminus can preventthe activation of an ethylene reporter construct in protoplasts fed with theethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Yanagi-sawa et al., 2003). This implies that the CTR1 N-terminus auto-inhibits theC-terminal kinase activity. Unlike Raf, however, removal of the CTR1 N-terminus does not elevate kinase activity in vitro. Additionally, plants car-rying a transgene for overexpression of the CTR1 kinase domain show noethylene insensitivity (Huang et al., 2003).

6.5.4 Other factors that potentially interact withand regulate CTR1 activity

Activation of the Raf carboxyl-terminal kinase domain is also regulated by de-phosphorylation, 14–3-3 binding and conformational changes. For example,it has been shown that 14–3-3 binds to the phosphorylated Ser259 in Raf-1and is required to stabilize the inhibitory self-interaction between the N-terminus and C-terminus (Fischer et al., 2009). Desphosphorylation of Ser259by PROTEIN PHOSPHATASE 2A (PP2A) will abolish 14–3-3 binding lead-ing to activation of the C-terminal kinase (Dhillon et al., 2002), whereas thiscan be reversed by the action of protein kinases (Zimmermann & Moelling,1999; Dumaz & Marais, 2003). A similar regulatory mechanism potentiallyexists for CTR1, since the N-terminus of Arabidopsis CTR1 can associate with14–3-3 proteins in the yeast two-hybrid assay (Chang et al., 1999), and aprotein-binding assay shows that Arabidopsis CTR1 can co-precipitate withthe C subunit of PP2A (Larsen & Cancel, 2003). In accordance with the Raf-activation model, disruption of PP2A function would prevent activation ofthe CTR1 kinase, which would lead to ethylene responses. Consistent withthis, a loss-of-function mutant of the PP2A catalytic subunit A displays anenhanced ethylene-response phenotype (Larsen & Cancel, 2003). It is cur-rently believed, however, that this phenotype is due to the role of PP2A inregulating ethylene biosynthesis, as shown by Muday et al. (2006).

The C-terminal kinase domain of Arabidopsis CTR1 can also associate withphosphatidic acid (PA) in vitro (Testerink et al., 2007). PA is the simplest phos-pholipid found in the cellular membrane and serves as a common precursorfor lipid biosynthesis. PA is also recognized as an important lipid messen-ger in cell growth, development and stress responses (Testerink & Munnik,2004). Raf-1 is one of the best-studied targets of PA. PA binds to the Raf-1kinase domain and is required for targeting Raf-1 to the plasma membrane(Ghosh et al., 1996). In plants, it has been shown that PA can inhibit CTR1



kinase activity and disrupt its interaction with the ethylene receptor ETR1in vitro, raising the possibility that PA negatively regulates CTR1 (Testerinket al., 2007). However, in vitro PA binding also disrupts the intra-molecularinteraction between the CTR1 N- and C-termini, which appears to promoteCTR1 activity. Therefore, additional studies are required to establish the effectof PA on CTR1 activity and localization in vivo.

6.6 Elusive targets of CTR1 signalling

In addition to understanding the regulation of CTR1, an important aim hasbeen to identify the immediate downstream targets of CTR1. Currently, theidentities of the direct effector(s) of CTR1 signalling remain unknown. Al-though EIN2 and EIN3 both act downstream of CTR1 in the ethylene-responsepathway (see Chapter 7), there is no evidence indicating that either proteinserves as a substrate for CTR1 kinase activity. Based on the sequence similar-ity of CTR1 to Raf-like kinases, it has long been expected that CTR1 acts as anMAPKKK. However, the identification of downstream MAPK componentshas evaded the standard approaches, perhaps due to issues of functionalredundancy and substrate specificity, yielding mutants that display eitherno phenotypes or pleiotropic phenotypes. Nevertheless, published reports ofMAPK cascades playing a role in ethylene signalling have appeared (Ecker,2004; Hahn & Harter, 2009; An et al., 2010), but such findings are under debateand the involvement of CTR1 in these pathways has not been established.

The possibility of an MAPK cascade in ethylene signalling was first raisedwhen an ethylene responsive MAPK-like activity was detected in Arabidopsisleaf extracts (Novikova et al., 2000). Subsequently, Ouaked et al., (2003) pro-posed that CTR1 acts in an MAPK module together with SIMKK (an MAPKK)and MPK6 (an MAPK). However, there was no biochemical evidence to con-nect CTR1 with SIMKK, and these findings remain in doubt amid variousconcerns (Ecker, 2004). More recently, an MAPKK–MAPK module consistingof MKK9–MPK3/MPK6, was proposed to stabilize the levels of EIN3 pro-tein via phosphorylation of EIN3 by MPK6 (Yoo et al., 2008; see Chapter 7).However, again there was no direct biochemical connection to CTR1. In fact,another group demonstrated that the MKK9–MPK3/MPK6 module elevatesethylene biosynthesis via the phosphorylation of two ACC synthases, whichaccumulate when phosphorylated by MPK6 (Liu & Zhang, 2004; Joo et al.,2008; Xu et al., 2008), a result that is also supported by Bethke et al. (2009).Consistent with this, An et al. (2010) presented genetic evidence that rules outthe possibility of MKK9 playing a role in the ethylene-signalling pathway.While it would not be unusual for the components of MAPK modules to berecruited to serve in multiple pathways with different targets, it is safe to saythat, to date, no MAPKKs or MAPKs have been confirmed as the targets ofCTR1 signalling.



6.7 CTR1 crosstalk and interactions with other signals

Because the loss of CTR1 function yields a plant with constitutive ethyleneresponses, ctr1 mutants have served a useful purpose in uncovering the rolesof ethylene in a variety of growth and developmental processes and havehelped to uncover interactions between ethylene-signalling and other path-ways. A good example is the delayed flowering phenotype of ctr1. This delayappears to be based on crosstalk between ethylene signalling and GA. GAis known to have a significant role in promoting the transition to flowering.Achard et al. (2007) showed that the delayed flowering in response to ethyleneis due, in part, to a reduction in bioactive GA levels. Accordingly, bioactiveGA levels were found to be reduced in the ctr1–1 mutant. A consequenceof reduced GA is the accumulation of DELLA-transcription factors, whichrepress GA responses such as the transition to flowering. ctr1–1, as well asthe ethylene-treated wild type, therefore exhibit delayed flowering due to anincrease in DELLA-transcription factors. Interestingly, treatment with exoge-nous GA not only rescues the delayed flowering in ctr1–1, but partly rescuesthe vegetative growth phenotypes of ctr1–1, such as the small rosette size andlonger petioles (Achard et al., 2007).

In common with ethylene-induced root hair differentiation in a numberof species, the Arabidopsis ctr1 mutant has a proliferation of root hairs thatform in ectopic locations in the root rather than in the strict pattern exhibitedby the wild type (Dolan et al., 1994). The ctr1btk mutant allele has a shorterroot with longer root hairs that develop at hyper-polar positions relative tothe wild type (Ikeda et al., 2009). Analysis of the ctr1btk mutant revealed thatCTR1 represses auxin biosynthesis in root tips, resulting in repression of theauxin concentration gradient (Ikeda et al., 2009). This led to the finding thatlocal auxin biosynthesis is responsible for gradient-directed planar polarityin the root.

Other examples of crosstalk between ethylene and other growth-regulatingsubstances have been revealed by the ctr1–1 mutant, including the follow-ing example. During germination and post-germination development, ctr1–1is more tolerant to salt and osmotic stress than the wild type, reflecting aninteraction between the ethylene- and abscisic acid (ABA)-signalling path-ways (Wang et al., 2007). ctr1–1 also has lower ascorbic acid content duringdark-induced leaf senescence (Gergoff et al., 2010) and exhibits inhibition oflateral root initiation and elongation (Negi et al., 2008). Mutants of ctr1 canalso bypass the need for GA in seed germination (Gibson et al., 2001), similarto ethylene overproducer mutants and ethylene-treated wild type plants. Theresponse of ctr1–1 seeds to imbibition has also connected ethylene signallingto the regulation of seed shape (Robert et al., 2008). ctr1 mutant alleles havealso been isolated in genetic screens for seedlings that are insensitive to theinhibitory effects of high levels of sucrose or glucose (Gibson et al., 2001;Leon & Sheen, 2003); this resistance indicates that ethylene signalling acts to



antagonize the response to high levels of sugar and implicates a connectionbetween sugar and stress signalling.

6.8 Conclusions

CTR1 is an important negative regulator in the ethylene-signalling path-way, and much has been elucidated regarding the CTR1 gene family and thesub-cellular localization and biochemical activity of the CTR1 protein. Yetcritical questions remain regarding CTR1 function in the pathway. In partic-ular, it is unclear how CTR1 is regulated and how it transduces its signal todownstream components. An underlying uncertainty amid these questionsis whether CTR1 functions as a Raf kinase. CTR1 is commonly referred toas a plant Raf (or Raf-like) kinase, and throughout this chapter intriguingparallels with Raf have been raised. To summarize these, CTR1 and Raf pos-sess similar enzymatic properties such as the ability to (auto)phosphorylateat Ser/Thr residues and accept MBP as a substrate (Huang et al., 2003). Inaddition, the emerging view of potentially complex mechanisms regulatingCTR1 activity bears remarkable similarities to the Raf model. Both proteinshave an N-terminus that is responsible for direct protein–protein interactionand subsequent membrane localization (Clark et al., 1998; Gao et al., 2003).The activation of both CTR1 and Raf requires protein interaction with an up-stream signalling component (ETR1 and Ras, respectively). Although thereis no sequence similarity between ETR1 and Ras, a structure for the ETR1receiver domain suggests that it may share a similar tertiary structure withthe Raf activator Ras (Chen et al., 1990). Additional proteins such as 14–3-3chaperones and phosphatases are involved in the intra-molecular interactionof Raf. Plant homologues of these proteins may also be associated with CTR1and ethylene signalling (Chang et al., 1999; Larsen & Cancel, 2003). It is thuscompelling to draw a parallel model for CTR1 based on the mammalian Raf.However, there is currently no biochemical or genetic evidence to supportthat CTR1 functions in vivo as a canonical Raf kinase.

CTR1, in a sense, is a familiar protein kinase in an unfamiliar setting.There are few known examples of a histidine kinase receptor that regulatesor interact directly with a Raf kinase. In addition, the regulatory N-terminusof Raf plays a critical role in repressing the carboxyl-terminal kinase do-main, whereas a similar function has not been demonstrated for the CTR1N-terminus (Huang et al., 2003). Most importantly, the phosphorylation sub-strate(s) of CTR1 has yet to be identified and the involvement of a MAPKcascade in ethylene signalling is still under debate (Ecker, 2004; Hahn &Harter, 2009). Thus, it is quite possible that CTR1 function has diverged soas not to fit the Raf paradigm. A scenario that remains to be tested is thedirect regulation by CTR1 of the next known downstream component, EIN2.If this proves to be the case, it would not be the first interesting twist in



the ethylene-response pathway. Thus, we anxiously await future discoveriesthat will shed light on this question as well as the mechanisms involved inregulating the CTR1 family.

Acknowledgements

The authors thank Zanetta Chang for assistance with Figure 6.1(a). SilinZhong would like to acknowledge the Human Frontier Science Program(LT000076/2009-L) and National Natural Science Foundation of China(NSFC30900783) for research support. Caren Chang would like to acknowl-edge the National Institutes of Health (1R01GM071855) and the NationalScience Foundation (MCB0923796) for research support.

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