Ethylene signaling: from mutants to molecules

353

The past decade has been incredibly productive for ethyleneresearchers. Major components in the ethylene signalingpathway in plants have been identified and characterized. Thepast year’s contributions include the crystallographic analysisof the Arabidopsis ETR1 receiver domain, antisense studiesof the tomato ethylene receptor genes LeETR4 and NR, andthe cloning and functional characterization of severalArabidopsis EREBP-related transcription activators andrepressors, and of an EIN3-ortholog of tobacco. Additionalevidence for the interconnection of the ethylene and auxinresponses was provided by the cloning and characterizationof Arabidopsis NPH4. Finally, the first discovery of ethyleneresponsiveness in an animal species implied a more universalrole for ethylene than previously thought.

AddressesPlant Biology Laboratory, The Salk Institute for Biological Studies,La Jolla, California 92037, USA

Current Opinion in Plant Biology 2000, 3:353–360

1369-5266/00/$ - see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsABA abscisic acid aux1 auxin1AXR1 AUXIN RESISTANT1CTR1 CONSTITUTIVE TRIPLE RESPONSE1EIL EIN3-like proteinEIN4 ETHYLENE INSENSITIVE4eir1 ethylene insensitive root1EREBP ETHYLENE RESPONSE ELEMENT BINDING PROTEINERF1 ETHYLENE RESPONSE FACTOR1ERS1 ETHYLENE RESPONSE SENSOR1ETR1 ETHYLENE RECEPTOR1hls1 hookless1ISR induced systemic resistanceMAP mitogen-activated proteinnph4 nonphototropic hypocotyl4NR NEVER RIPEPst Pseudomonas syringae pv. tomatoRAN1 RESPONSIVE TO ANTAGONIST1SAR systemic acquired resistanceTEBS TEIL-binding siteTEIL TOBACCO EIN3-LIKETMV tobacco mosaic virus

IntroductionEthylene is an endogenous plant hormone that affectsmany aspects of growth and development, such as germi-nation, flower and leaf senescence, fruit ripening, leafabscission, cell-fate determination in the root epidermis,root nodulation, sex determination, programmed cell death,and responsiveness to stress and pathogen attack [1,2].Elucidation of the mechanism of ethylene perception andsignal transduction began with the isolation of mutants thathave defective ethylene responses. Arabidopsis seedlingsgrown for three days in the dark under continuous exposureto ethylene exhibit a phenotype collectively known as the

‘triple response’. This phenotype includes thick and shortroots and hypocotyls, and an exaggerated curvature of theapical hook. Screens for mutants that are unable to gener-ate the triple response when treated with ethylene havebeen used to identify ethylene insensitive mutants.Similarly, plants that exhibit the triple response in theabsence of exogenous ethylene have been identified aseither ethylene-overproducing or constitutive-signalingmutants. Overall, more than a dozen genes have beenimplicated in the ethylene-signaling pathway, and theirorder of action has been tentatively determined using acombination of genetic and molecular approaches(reviewed in [2]).

Ethylene is perceived by a family of integral membranereceptors. In Arabidopsis, at least five family members areinvolved: ETHYLENE RECEPTOR1 (ETR1), ETR2,ETHYLENE INSENSITIVE4 (EIN4), ETHYLENERESPONSE SENSOR1 (ERS1), and ERS2 [3–6].Ethylene binds to the receptors via a copper cofactor [7••],and genetic studies suggest that hormone binding inacti-vates the receptors [8,9••]. In the absence of ethylene, thereceptors are predicted to be functionally active histidine-kinases that activate a Raf-like serine/threonine kinase,CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), also anegative regulator of the pathway [10]. Genetic studies alsopredict that EIN2, EIN3, EIN5, and EIN6 [11] are positiveregulators of the ethylene response. It is not yet known howthe ethylene signal is transduced via these molecules at thebiochemical level. EIN2 is a metal-ion transporter-relatedintegral-membrane protein, whose function is not well-understood [12••]. EIN5 and EIN6 are proteins ofunknown sequence and function, which are currentlyunder investigation. The nuclear protein EIN3 and its par-alogs, the EIN3-like proteins (EILs), are transcriptionfactors that bind to the promoters of ethylene-responsegenes such as ETHYLENE RESPONSE FACTOR1 (ERF1)and initiate a transcriptional cascade leading to the regula-tion of ethylene target genes [13,14]. In this review, wedescribe recent advances in the field of ethylene signaltransduction in the context of the previously defined com-ponents of the pathway. The focus is on Arabidopsis as thisis the plant in which the majority of signaling componentshave been identified.

Ethylene perception and signal transductionGenetic epistasis studies of Arabidopsis signaling mutantsrevealed that ETR1, ETR2 and EIN4, along with theirhomologues ERS1 and ERS2, work upstream of CTR1,whereas EIN2, EIN3, EIN5 and EIN6 work downstream(reviewed in [2]). Detailed analysis of these mutants, aswell as the recent cloning and characterization of some ofthe corresponding genes, now allows us a glimpse of thecomplex nature of the ethylene signaling pathway in plants.

Ethylene signaling: from mutants to moleculesAnna N Stepanova and Joseph R Ecker

The Arabidopsis ethylene-receptor family is comprised offive membrane proteins (i.e. ETR1, ETR2, EIN4, ERS1,and ERS2) that are similar to the two-component histidine-kinase regulators found in bacteria and fungi [3–6].Two-component systems generally consist of a sensor molecule with a histidine-kinase domain that autophospho-rylates in response to an environmental stimulus, and aresponse regulator with a receiver domain that accepts thephosphate from the histidine residue of the sensor on itsaspartate residue [15]. In ETR1, ETR2, and EIN4, fea-tures of both a sensor and a response-regulator arecombined in the same molecule [3,5,6]. In contrast, ERS1and ERS2 lack the receiver domain, implying that these

molecules either utilize the receiver of ETR1, ETR2 orEIN4, or employ other response regulators [4,6]. In fact,several molecules with homology to bacterial receiver pro-teins are known [16], although there is no evidence yet thatthey are involved in ethylene signal transduction.

The transmembrane-spanning domain of the ethylenereceptors is located in the amino terminus, followed by aGAF domain, and a histidine-kinase domain. In some ofthe receptors (i.e. ETR1, ETR2 and EIN4) a receiverdomain is found in the carboxyl terminus [6]. On the basisof sequence similarities, irrespective of the presence of thereceiver domain, the receptor family can be subdivided into

354 Cell signalling and gene regulation

Figure 1

A model for ethylene signaling in Arabidopsis.(a) Ethylene binds via a copper co-factor toplasma-membrane-embedded hydrophobicpockets formed by the amino termini (N) ofthe ETR1, ETR2, EIN4, ERS1 and ERS2homodimers. Ethylene binding deactivates thereceptor molecules and, in the absence of apositive regulatory signal from the receptors,the CTR1 protein becomes inactive. Furthertransduction of the ethylene signal requiresthe positive regulators EIN2, EIN3, EIN5, andEIN6 that function downstream of CTR1.(b) EIN2 is an integral-membrane protein ofunknown subcellular localization.(c) Downstream of EIN2 is the EIN3 family oftranscription factors, these bind to the EBSelement in the promoter of the ERF1 geneand activate its expression in an ethylene-dependent manner. (d) ERF1, in turn,interacts with the GCC-box of ethylene-response genes and presumably turns them‘on’. Other EREBPs, which function asethylene-regulated transcriptional activators orrepressors, are also likely to work downstreamof the EIN3 protein family. Signal transductionfrom CTR1 may involve a MAPK cascade. Theorder of action of EIN5 and EIN6 is unknown.Modular structures of the cloned signalingcomponents are shown. Gray shadingindicates known protein–protein interactions.‘?’ represents a novel or unknown compound.C, carboxy terminal; DB, DNA binding; G,GAF; HK, histidine kinase; N, amino terminal;R, receiver; STK, serine/threonine kinase andTM, transmembrane. DNA elements: EBS,EIN3-binding site; and GCC, GCC-box.

EIN5EIN6?

CuCu

RC

HK

NN

C

CuCu

NN

CC

N

C

NC

?

OtherEREBPs

?

EBS ERF1

DB DB

EIN3

EILs ?

GCC

ERF1

?

DB

? ? N

C

N

C

Ethylene responses

Target gene

STK

ERS1ERS2

CTR1

ETR1ETR2EIN4

MAPKKMAPK?

Membrane

Membrane

?

?

EIN2

HKHKHK

R

?

?

G GG G

Cytoplasm

Nucleus

Current Opinion in Plant Biology

TM

TM

C2H4

(a)

(b)

(c)

(d)

two classes. Members of the first class, ETR1 and ERS1, pos-sess three hydrophobic subdomains in the amino terminus,and a well-conserved histidine-kinase domain in the carboxy-terminal part of the protein. In contrast, members of thesecond class, ETR2, EIN4 and ERS2, possess four stretchesof hydrophobic amino acids, and lack some of the hallmarksof the bacterial histidine kinases [6]. Interestingly, represen-tatives of both classes of ethylene receptors have beenidentified in other plant species, implying that the mecha-nism of ethylene perception is conserved in flowering plants(reviewed in [17]). Functional equivalence of the two classesof receptor molecules is suggested by the antisense studies ofthe tomato NEVER RIPE (NR) (which encodes a class 1member, with no receiver domain) and LeETR4 (whichencodes a class 2 member, with a receiver domain) receptorgenes [18••]. Reduction in the level of LeETR4 mRNA leadsto the enhanced ethylene sensitivity and responsiveness oftomato plants, but not in the lines that also overexpress NR.Similarly, no phenotype is observed in transgenic lines har-boring the NR antisense construct, as the endogenous levelsof LeETR4 mRNA are upregulated in these plants and presumably compensate for the loss of NR [18••].

The conclusion that ETR1 and its homologues are, in fact,ethylene receptors is based on several observations. Inaddition to the similarity of these proteins to two-compo-nent sensors and regulators, dominant etr1 mutants bindless ethylene than do wild-type plants [19]. Heterologousexpression of ETR1 or ERS1 in yeast confers the ability tobind ethylene with high affinity [7••,20]. Moreover, severalmutant versions of ETR1, which result in dominant ethyl-ene insensitivity in plants, have reduced or no ability tobind ethylene when expressed in yeast [20,21•].

Both in yeast and in planta, the ETR1 protein forms mem-brane-associated disulfide-linked homodimers [22], butcovalent linkage of the monomers is not essential for ethyl-ene binding [20]. Binding of ethylene is accomplished by ahydrophobic membrane-localized pocket in the amino-ter-minal part of ETR1 [20,21•] and requires a copper ion as acofactor [7••]. Copper ions are thought to be supplied to thereceptors by RESPONSIVE TO ANTAGONIST1(RAN1), a putative Arabidopsis copper-transporting P-typeATPase homologous to the yeast Ccc2p and humanMenkes/Wilson disease proteins [9••]. Co-suppression ofRAN1 in transgenic lines, as well as a hypomorphic ran1-3allele, results in plants with a constitutive triple-responsephenotype that can be partially rescued by exogenous cop-per application [9••,23•]. Two missense alleles of RAN1, oneharboring a mutation in a predicted phosphatase domain(ran1-1) and the other in the metal-binding domain (ran1-2),are predicted to result in a relaxed or altered ligand speci-ficity. These mutants have a wild-type morphology in airand ethylene but, unlike wild-type plants, also exhibit thetriple response when treated with the receptor antagonisttrans-cyclooctene [9••]. Recent studies of a severe ran1-3allele suggest a more general requirement for RAN1 in plantgrowth and development [23•].

The cytoplasmic receiver domain of ETR1 has beenrecently crystallized [24,25•]. In contrast with earlierresults, which suggested that the membrane-localizedamino-terminal domain of ETR1 is necessary and suffi-cient for ETR1 homodimerization in yeast [22], theE. coli-expressed cytoplasmic carboxyl terminus of ETR1exists in a dimeric form, both in crystals and in solu-tion [25•]. This discrepancy may be explained by possibledifferences in the phosphorylation states of E. coli- versusyeast-expressed ETR1 proteins. Phosphorylation has beenshown to promote the in vivo homodimerization of thereceiver domains of the E. coli proteins NtrC andPhoB [26], and to repress the heterodimerization of theresponse regulator CheY with its cognate histidine-kinaseCheA [27,28]. To date, no in planta phosphorylation/dimer-ization studies have been performed on the receptormolecules, and only the histidine-kinase autophospho-rylation of a yeast-expressed Arabidopsis ETR1histidine-kinase domain has been reported [29].

Analysis of the ETR1 receiver-domain crystals reveals thatthe β-sheet dimer interface is similar to that found in sev-eral bacterial response regulators and is provided by thecarboxyl termini of the monomers. In contrast, the archi-tecture of the predicted active site of the ETR1 receiver isunique [25•]. It remains to be determined whether theETR1 receiver can indeed accept a phosphate from thehistidine-kinase of the sensor domain, and if it does, whatthe next component of the phosphorelay is. One likely can-didate is the Raf-like kinase CTR1. This protein is capableof interacting with the cytoplasmic portions of ETR1 andERS1 both in vitro and in yeast cells [30]. Null mutationsin the CTR1 gene result in a constitutive triple-responsephenotype, indicating that CTR1 is a negative regulator ofthe ethylene signaling pathway [10]. Unlike the ctr1mutant, null receptor mutants do not display ethylene phe-notypes [8]. The functional redundancy of the receptors is,however, unmasked in the triple and quadruple receptormutants which possess a strong ctr1-like morphology [8]. Intomato, down-regulation of just one of the five knownreceptor genes, LeETR4, leads to enhanced ethyleneresponsiveness [18••]. These results unambiguously showthat the receptors function as negative regulators of ethyl-ene signaling in a variety of plants. The dominant nature ofthe ethylene-insensitive receptor mutations may beexplained by one of several possibilities [21•]. Constitutiveactivation of these proteins may be caused by either theirabolished/reduced ability to bind ethylene (as shown foretr1-1, etr1-3 and etr1-4) or the constitutive activation of thekinase or receiver domain, such that they are no longercapable of responding to the negative regulatory signal ofethylene binding (as predicted for etr1-2) [21•].

CTR1, which has been shown genetically to functiondownstream of the receptors, is homologous to the Raffamily of serine/threonine protein kinases [10]. Consistentwith the role of Raf kinases in mammals, CTR1 may be amitogen-activated protein (MAP) kinase kinase kinase

Ethylene signaling Stepanova and Ecker 355

(MAPKKK), and the repression of ethylene signaling mayoccur via a MAP kinase kinase (MAPKK) and a MAPkinase (MAPK). Although this is certainly a possibility, anda number of MAPKKs and MAPKs have been identified inplants [2], no biochemical evidence currently exists toprove that a MAPK cascade plays a role in ethylene signaltransduction. Identification of knockout mutations in thegenes encoding these kinases should resolve this issue, butto date, no direct biochemical connection has been madebetween CTR1 and the downstream positive regulators ofethylene signaling EIN2, EIN3, EIN5, EIN6, and ERF1.

EIN2 encodes a novel integral membrane protein withsequence similarity to a mammalian family of NRAMPmetal-ion transporters [12••]. This homology is limited tothe transmembrane amino-terminal part of the protein.The extended carboxyl terminus is novel, with the excep-tion of a coiled-coil motif that has been implicated inprotein–protein interactions in other organisms [12••]. Thefunction of EIN2 in ethylene signaling is not understood.By analogy with the NRAMP proteins, the transmembraneportion of EIN2 has been hypothesized to function as atransporter of divalent cations [12••]. To date, however, nometal-binding or metal-transporting properties of EIN2have been observed. Both the amino-terminal and the car-boxy-terminal domains of EIN2 are essential for thefunction of this protein: truncated versions of EIN2 areunable to complement the ethylene insensitivity of the ein2mutants [12••]. Interestingly, overexpression of the car-boxy-terminal part of EIN2 in the ein2 mutant backgroundtriggers the partial activation of ethylene responses andrestores the ability of the mutant to respond to paraquat andjasmonic acid, but not to ethylene [12••]. These results sug-gest that the amino-terminal part of EIN2 is required forsensing the ethylene signal from the upstream signalingmolecules, whereas the carboxy-terminal part is necessaryand sufficient for transducing this signal to the downstreamcomponents of the ethylene pathway.

Nuclear eventsGenetic data suggest that EIN3 acts downstream ofEIN2 [12••,13]. EIN3 is a novel nuclear-localized proteinwith DNA-binding properties [13,14]. It is thought to serveas a transcription factor that, in response to the ethylenesignal, binds to specific sequences in the promoters of tar-get genes and activates their transcription [14]. To date, theethylene-inducible gene ERF1 is the only known directtarget of Arabidopsis EIN3. EIN3 dimers interact with aunique imperfect palindromic repeat element in the pro-moter of ERF1 [14]. Homodimers of two EIN3 paralogs,EIL1 and EIL2, are also capable of binding to this DNAsequence in vitro [14], but no heterodimerization betweenthese proteins and EIN3 has been reported.

EIN3-like transcription factors are not unique toArabidopsis. Recently an ortholog of EIN3/EIL has beencloned from tobacco and has been implicated in ethylenesignaling by overexpression studies in Arabidopsis [31••].

TOBACCO EIN3-LIKE (TEIL) protein is 92% identicalto EIN3 in the deduced DNA-binding domain, suggestingthat these proteins may bind to the same DNA sequence[31••]. A random site-selection approach using an E. coli-expressed TEIL protein yielded a non-palindromicsequence TEIL-binding site (TEBS) that is very similar tothe partial EIN3-binding site from the ERF1 promoter.Importantly, this element was shown to be sufficient for theTEIL-mediated transcriptional activation of a TEBS-drivenreporter [31••], providing additional evidence that EIN3-related proteins are indeed activators of transcription.

The immediate target of Arabidopsis EIN3, ERF1,encodes a protein that belongs to the ETHYLENERESPONSE ELEMENT BINDING PROTEIN(EREBP) family of DNA-binding proteins and that iscapable of binding to the GCC-box [14]. The GCC-box isa cis-element found in the promoters of several pathogen-related genes, many of which are ethylene responsive.ERF1 is thought to function as a transcription factor, thusimplying that a transcriptional cascade is involved in eth-ylene signaling. ERF1, like EIN3, is a positive regulatorof ethylene signaling. In fact, overexpression of ERF1leads to the constitutive activation of many ethyleneresponses. The triple response of ERF1-overexpressingplants is, however, incomplete, suggesting that other fac-tors may be required for a full response [14]. Indeed, alarge number of EREBP genes exists in Arabidopsis andother species [32], but the expression of only a fraction ofthem is regulated by ethylene [33–35,36••,37••]. In tomato,the product of an ethylene-inducible EREBP, PTI4, can bephosphorylated by the serine/threonine kinase PTO, andthe resulting phosphorylated version of PTI4 has a greateraffinity for the GCC-box than does unphosphorylatedPTI4 [36••]. Consistent with this observation, the con-comitant overexpression of PTO and PTI4 in tomato leadsto the induction of pathogenesis-related (PR) genes thathave a GCC-box in their promoters [36••].

Not all ethylene-responsive EREBPs encode transcrip-tional activators: some encode repressors of transcription[37••]. Given the myriad of responses to ethylene, itseems logical to expect that some ethylene-related phe-notypes result from both the activation of some genes andrepression of others. Furthermore, a number of ethylene-responsive genes possess neither the EIN3-bindingelement nor the GCC-box in their promoters. This obser-vation indicates that transcription factors that areunrelated to either EIN3 or ERF1, and which have a dif-ferent binding specificity, may also be involved in thelater steps of ethylene signaling. Identification of thesemolecules will be crucial to complete our understandingof ethylene-mediated gene regulation.

Unraveling of the web of hormone interactions:ethylene and auxinThe cloning of genes identified in genetic screens for plantsaffected in the ‘triple response’ has led to a tremendous


improvement in our understanding of ethylene signalingprocesses. Although the elucidation of the intersectionbetween the ethylene and other hormone signaling path-ways is in its infancy, recent studies of several auxin mutantsshow promise for unraveling this ‘web’ of interactions.

Mutations in the Arabidopsis gene AUXIN RESISTANT1(AXR1) are characterized not only by auxin insensitivity,but also by reduced sensitivity to ethylene in seedlingroots and apical hooks [38–40]. Cloning of AXR1 revealedsequence similarity to the ubiquitin-activating enzymeE1 [41], and recent genetic and biochemical findings sug-gest that AXR1 may indeed be involved inubiquitin-mediated protein degradation [42,43]. Theauxin mutants ethylene insensitive root1 (eir1) and auxin1(aux1) are selectively resistant to ethylene in the seedlingroot [11,44]. AUX1 and EIR1 encode proteins withhomologies to plant and fungal amino-acid permeases, andbacterial transporters. They are thought to function inpolar auxin transport as influx and efflux carriers, respec-tively [45,46•,47–50]. hookless1 (hls1) mutants are deficientin the formation of the apical hook both in air and in eth-ylene because of an inability to undergo differential cellelongation in that part of the seedling [40,51]. The HLS1gene possesses a GCC-box in its promoter and is ethyleneinducible [40], implicating the EREBP family of transcrip-tion factors in its regulation. HLS1 encodes atransacetylase-like molecule [40] whose biochemical func-tion has yet to be demonstrated. Interestingly, thenonphototropic hypocotyl4 (nph4) mutant is impaired inauxin-induced hypocotyl bending, but this defect can befunctionally complemented by the exogenous applicationof ethylene [52•]. The NPH4 gene product, ARF7, belongsto the family of auxin-response element DNA-bindingproteins. Thus, in the nph4 mutant, ethylene may enhanceauxin responsiveness by stimulating either the activity orthe sensitivity of a redundant ARF system [52•].

Ethylene and disease resistanceEthylene gas is released upon pathogen infection and isthought to be a part of the plant defense mechanismagainst the spread of pathogens. In the past year, severalstudies have demonstrated that a functional ethylene sig-naling pathway is required for resistance against some, butnot all, pathogens. EIN2 was shown to be essential forpathogen-mediated systemic induction of the basic chitinasePR-3 and a hevein-like gene PR-4 in Arabidopsis upon infec-tion with the fungus Alternaria brassicicola [53•]. Localinduction of the HEL, CHIB and PDF1.2 genes by a cul-ture filtrate from the virulent Gram-negative bacteriumErwinia carotovora subsp. carotovora was also severelyreduced in the ein2-1 and etr1-1 mutants [54•].Furthermore, ein2-1 plants exhibited greater susceptibilityto infection by E. carotovora subsp. carotovora [54•] and thefungus Botrytis cinerea, but not to infection by avirulentstrains of the fungi A. brassicicola and Peronospora parasit-ica [53•]. Systemic acquired resistance (SAR) to a virulentbacterial strain of Pseudomonas syringae pv. tomato (Pst)

following preinoculation with an avirulent strain of Pst wasunaffected in all of the ethylene-insensitive mutants tested(i.e. etr1-1, ein2-1, ein3-1, ein4-1, ein5-1, and ein6) [55•].Similarly, the etr1-1 and etr1-3 mutants exhibited a normalSAR response to P. parasitica infection upon pretreatmentwith the product of the hrpN gene of Erwinia amylovora[56•]. In contrast, induced systemic resistance (ISR)against virulent Pst upon preliminary root colonization withthe nonpathogenic rhizobacterium Pseudomonas fluorescenswas completely abolished in all of the tested ethylene-insensitive mutants (i.e. etr1-1, ein2-1, ein3-1, ein4-1, ein5-1,and ein6) [55•]. Interestingly, under these conditions, noISR was observed in eir1-1 and axr1-12 mutants, which areselectively insensitive to ethylene in the root.Furthermore, in the eir1-1 mutant, the ISR was abolishedonly upon preinoculation of the rhizobacterium in the rootand not in the leaves, suggesting that the ISR requiresintact ethylene signaling at the site of preinoculation [55•].

In tobacco, necrotic lesion formation and induction of basicPR gene expression upon infection with tobacco mosaicvirus (TMV) were also shown to require ethylene [57•].Application of ethylene biosynthesis and action inhibitorssignificantly reduced the severity of lesions and sup-pressed the induction of the pathogen-related genes basicPR1 and P1-II, but not of acidic PR1 [57•]. Ethylene over-production in tobacco resulted in enhanced lesionformation upon TMV inoculation [57•]. Conversely, ethyl-ene insensitivity had no effect on the hypersensitiveresponse or the overall resistance of Arabidopsis plants toturnip crinkle virus infection [58]. In tomato, a virulentbacterial strain of Xanthomonas campestris pv. vesicatoriacaused milder symptoms (i.e. chlorosis and necrosis) in theethylene insensitive Nr mutant and NR-overexpressingplants than in wild-type plants, in spite of similar bacterialtiters [59•]. This increased plant tolerance was attributedto reduced ethylene sensitivity. Both wild-type and ethyl-ene-insensitive Nr and NR-overexpressors were, however,equally resistant to an avirulent strain of X. campestris pv.vesicatoria [59•]. Although the important role of ethylene inplant disease resistance is evident, this hormone seems toaffect separate aspects of plant–pathogen interactions indifferent ways, depending on the host and pathogenspecies, and the conditions tested.

Ethylene response in animalsThe possession of an ethylene signal transduction pathwayis not unique to Arabidopsis. Orthologs of all of the majorsignaling components known to be involved in thisArabidopsis pathway have been identified in several otherplant species (reviewed in [2,17]; JR Ecker, unpublishedresults). Moreover, a bacterial protein that has bothsequence homology to the transmembrane domain ofETR1 and ethylene binding properties has been isolatedfrom Synechocystis sp. [7••]. Recently, an animal species,Suberites domuncula, has been shown for the first time, torespond to ethylene, both physiologically and at the mole-cular level [60••]. In this sponge, ethylene can repress


starvation-induced apoptotic cell death, and the mRNAlevels of at least two genes, SDERR and CaM kinase II, areupregulated as a result of ethylene exposure [60••].Although it is not yet clear whether this animal can senseand respond to ethylene gas via a conventional ‘plant-spe-cific’ pathway, the fact that gene expression is affectedsuggests the existence of some sort of perception andtransduction pathway for this gas signal.

ConclusionsAlthough the past decade has seen significant advancestoward understanding ethylene action in plants, manyquestions still remain to be answered. In the short term,the functional analysis of known regulatory and signalingcomponents as well as the cloning and characterization ofadditional ethylene pathway genes, such as EIN5 andEIN6, will certainly provide new insight into the mysteriesof ethylene metabolism, perception and transduction. Thenext exciting challenge will be to interconnect the differ-ent hormone signaling pathways in an attempt tounderstand how a plant generates a balanced response to amultitude of growth promoting and inhibitory stimuli.

UpdateNew alleles of ein2 and ctr1 have recently been isolatedin screens for mutants affected in their response toabscisic acid (ABA) [61,62]. The results of the analysisof these well-characterized ethylene mutants in ABA-response assays suggest that both ethylene and ABAaffect the same developmental processes. Whereas theregulation of seed dormancy/germination and root elon-gation by ABA requires intact ethylene signaling, thecontribution of each of the two hormonal pathways isdifferent in these two processes [61,62].

AcknowledgementsWe would like to thank Jose Alonso and Robert McGrath for theircomments on this manuscript. Research in our laboratory is supported bythe National Science Foundation and the US Department of Energy.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest•• of outstanding interest

1. Abeles FB, Morgan PW, Saltveit ME Jr: Ethylene in Plant Biology,2nd edn. New York: Academic Press; 1992.

2. Johnson PR, Ecker JR: The ethylene gas signal transductionpathway: a molecular perspective. Annu Rev Genet 1998,32:227-254.

3. Chang C, Kwok SF, Bleecker AB, Meyerowitz EM: Arabidopsisethylene-response gene ETR1: similarity of product to two-component regulators. Science 1993, 262:539-544.

4. Hua J, Chang C, Sun Q, Meyerowitz E: Ethylene insensitivityconferred by Arabidopsis ERS gene. Science 1995,269:1712-1714.

5. Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB,Meyerowitz EM: ETR2 is an ETR1-like gene involved in ethylenesignaling in Arabidopsis. Proc Natl Acad Sci USA 1998,95:5812-5817.

6. Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR,Meyerowitz EM: EIN4 and ERS2 are members of the putative

ethylene receptor gene family in Arabidopsis. Plant Cell 1998,10:1321-1332.

7. Rodriguez FI, Esch JJ, Hall AE, Binder BM, Schaller GE, Bleecker AB: •• A copper cofactor for the ethylene receptor ETR1 from

Arabidopsis. Science 1999, 283:996-998.Ethylene binds hydrophobic membrane-localized pockets formed by theamino terminus of ETR1 via a copper cofactor. The amino-terminal domainsof ETR1 and Etr1-1 (as identified in the etr1-1 ethylene insensitive mutant)were expressed in a heterologous yeast system. Copper ions were shown toco-purify with the wild-type ETR1 protein, but not with its mutant version. Asequence homologous to ETR1 was identified in the genome of thecyanobacterium Synechocystis sp. and was shown to encode a functionalethylene-binding protein.

8. Hua J, Meyerowitz EM: Ethylene responses are negativelyregulated by a receptor gene family in Arabidopsis thaliana. Cell1998, 94:261-271.

9. Hirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, •• Nourizadeh S, Alonso JM, Dailey WP, Dancis A, Ecker JR:

Responsive-to-antagonist1, a Menkes/Wilson disease-relatedcopper transporter, is required for ethylene signaling inArabidopsis. Cell 1999, 97:383-393.

Two alleles of a novel ethylene mutant, ran1, were identified. These mutantsdisplay the triple response not only in the presence of ethylene, but also whenexposed to the ethylene-receptor antagonist trans-cyclooctene. Molecularcloning of the RAN1 gene revealed its homology to copper-ion-transportersfrom yeast and humans. The RAN1 cDNA was able to complement a yeastmutant that lacks a functional RAN1 ortholog, CCC2. The involvement ofRAN1 in copper trafficking in plants was implied by the ability of exogenouscopper to partially rescue the ran1 mutant phenotype. Transgenic plants thatco-suppress RAN1 displayed constitutive ethylene phenotypes. These resultssuggest that RAN1 is necessary for delivering copper ions to other ethylenesignaling components, presumably the receptors.

10. Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR: CTR1, anegative regulator of the ethylene response pathway inArabidopsis, encodes a member of Raf family of protein kinases.Cell 1993, 72:427-551.

11. Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR: Geneticanalysis of ethylene signal transduction in Arabidopsis thaliana:five novel mutant loci integrated into a stress response pathway.Genetics 1995, 139:1393-1409.

12. Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR: EIN2, a •• bifunctional transducer of ethylene and stress responses in

Arabidopsis. Science 1999, 284:2148-2152.The EIN2 gene was cloned using a map-based approach. It was found toencode a protein with a novel carboxy-terminal hydrophilic domain and ahydrophobic amino terminus, similar to the NRAMP metal-ion transporters.Transgenic studies in Arabidopsis showed that overexpression of the carboxyterminus in the ein2 mutant background results in a constitutive activation ofethylene responses. Transgenic lines were as insensitive to ethylene gas as theein2 mutant alone, indicating the requirement of the amino terminus of EIN2for sensing ethylene. Transgene-mediated partial activation of the downstreamsignaling events depended on functional EIN3, and was sufficient to restorethe ability of the ein2 mutant to respond to paraquat and jasmonate.

13. Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR:Activation of the ethylene gas response pathway in Arabidopsisby the nuclear protein ETHYLENE-INSENSITIVE3 and relatedproteins. Cell 1997, 89:1133-1144.

14. Solano R, Stepanova A, Chao Q, Ecker JR: Nuclear events inethylene signaling: a transcriptional cascade mediated byETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 1998, 12:3703-3714.

15. Pirrung MC: Histidine kinases and two-component signaltransduction systems. Chem Biol 1999, 6:167-175.

16. Imamura A, Hanaki N, Nakamura A, Suzuki T, Taniguchi M, Kiba T,Ueguchi C, Sugiyama T, Mizuno T: Compilation and characterizationof Arabidopsis thaliana response regulators implicated in His-Aspphosphorelay signal transduction. Plant Cell Physiol 1999,40:733-742.

17. Chang C, Shockey JA: The ethylene-response pathway: signalperception to gene regulation. Curr Opin Plant Biol 1999,2:352-358.

18. Tieman DM, Taylor MG, Ciardi JA, Klee H: The tomato ethylene •• receptors NR and LeETR4 are negative regulators of ethylene

response and exhibit functional compensation within a multigenefamily. Proc Natl Acad Sci USA 2000, 97:5663-5668.

An antisense strategy was used to examine the role of two ETR1 orthologsfrom tomato, NR and LeETR4. Reducing the level of LeETR4 mRNA caused


partial activation of ethylene signaling and enhanced sensitivity to ethylenegas, indicating the negative regulatory role of this gene in the ethylene path-way. Overexpression of the NR transgene in LeETR4-antisense lines sup-pressed ethylene hypersensitivity, implying that the two receptor proteins NRand LeETR4 are functionally equivalent. Antisense-elicited reduction in theNR message level was paralleled by an endogenous increase in the level ofLeETR4 mRNA, resulting in normal ethylene responses.

19. Bleecker AB, Estelle MA, Somerville C, Kende H: Insensitivity toethylene conferred by a dominant mutation in Arabidopsisthaliana. Science 1988 241:1086-1089.

20. Schaller GE, Bleecker AB: Ethylene-binding sites generated inyeast expressing the Arabidopsis ETR1 gene. Science 1995,270:1809-1811.

21. Hall AE, Chen QHG, Findell JL, Schaller GE, Bleecker AB: The • relationship between ethylene binding and dominant insensitivity

conferred by mutant forms of the ETR1 ethylene receptor. PlantPhysiol 1999, 121:291-299.

Several mutant alleles of ETR1 were examined for the ability of their encodedproteins to bind ethylene. The mutant proteins fell into two categories. First,yeast-expressed ETR1-1, ETR1-3 and ETR1-4 proteins had a reduced or nullability to bind ethylene. Second, ETR1-2 bound ethylene gas to the sameextent as the wild-type ETR1 protein. Therefore, ethylene insensitivity mayarise from the inability of the receptor molecules to either perceive ethyleneor transduce the signal from the sensor to the output domain.

22. Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB:The ethylene response mediator ETR1 from Arabidopsis forms adisulfide-linked dimer. J Biol Chem 1995, 270:12526-12530.

23. Woeste KE, Kieber JJ: A strong loss-of-function mutation in RAN1• results in constitutive activation of the ethylene response

pathway as well as a rosette-lethal phenotype. Plant Cell 2000,12:443-455.

The strong allele of the RAN1 gene, ran1-3, was identified and character-ized. The ran1-3 mutant exhibits constitutive ethylene morphologies that aresimilar to those of the ctr1 mutant and of RAN1-cosuppressed lines, but aremore severe and characterized by rosette-lethality. Double-mutant analysisconfirmed that RAN1 occupies an early position in the ethylene signalingpathway. The rosette-lethal phenotype of ran1-3 is thought to be unrelatedto ethylene as it cannot be suppressed in the Ein mutants.

24. Grantz AA, Muller-Dieckmann HJ, Kim SH: Subcloning,crystallization and preliminary X-ray analysis of the signal receiverdomain of ETR1, an ethylene receptor from Arabidopsis thaliana.Acta Crystollogr 1998, 54:690-692.

25. Muller-Dieckmann HJ, Grantz AA, Kim SH: The structure of the • signal receiver domain of the Arabidopsis thaliana ethylene

receptor ETR1. Struct Fold Des 1999, 7:1547-1556.The crystal structure of the ETR1 receiver domain (amino acids 604–738) isdescribed. This polypeptide forms dimers in crystals and in solution. Thestructure of the ETR1 monomer is similar to that of several bacterialresponse regulators.

26. Fiedler U, Weiss V: A common switch in activation of the responseregulators NtrC and PhoB — phosphorylation inducesdimerization of the receiver modules. EMBO J 1995,14:3696-3705.

27. Falke JJ, Bass RB, Butler SL, Chervitz SA, Danielson MA: The two-component signaling pathway of bacterial chemotaxis: amolecular view of signal transduction by receptors, kinases, andadaptation enzymes. Annu Rev Cell Dev Biol 1997, 13:457-512.

28. Welch M, Chinardet N, Mourey L, Birck C, Samama JP: Structure ofthe CheY-binding domain of histidine-kinase CheA in complexwith CheY. Nat Struct Biol 1998, 5:25-29.

29. Gamble RL, Coonfield ML, Schaller GE: Histidine kinase activity ofthe ETR1 ethylene receptor from Arabidopsis. Proc Natl Acad SciUSA 1998, 95:7825-7829.

30. Clark KL, Larsen PB, Wang XX, Chang C: Association of theArabidopsis CTR1 Raf-like kinase with the ETR1 and ERSethylene receptors. Proc Natl Acad Sci USA 1998, 95:5401-5406.

31. Kosugi S, Ohashi Y: Cloning and DNA-binding properties of a •• tobacco Ethylene-Insensitive3 (EIN3) homolog. Nucleic Acids Res

2000, 28:960-967.The tobacco EIN3-related gene TEIL encodes a protein with 60% and35–80% amino-acid identity to Arabidopsis EIN3 and EILs, respectively.Overexpresssion of TEIL in Arabidopsis resulted in a constitutive activationof ethylene responses. A random-site selection approach was employed toidentify a DNA sequence, TEBS, to which the TEIL protein binds in vitro.The consensus sequence resembles a half-site of the Arabidopsis EIN3binding box found in the ERF1 promoter. Transient expression of a reporter

construct that harbors four copies of TEBS upstream of a minimal promoterdemonstrated the sufficiency of this sequence to support the activation ofTEIL-mediated transcription. Furthermore, increased TEBS-binding activitieswere detected in the nuclear extracts prepared from the leaves of ethylene-treated plants. The DNA-binding domain of TEIL was mapped to the amino-terminal part of the protein and was found to be remarkably similar to that ofthe Arabidopsis orthologs.

32. Riechmann JL, Meyerowitz EM: The AP2/EREBP family of planttranscription factors. J Biol Chem 1998, 379:633-646.

33. Ohme-Takagi M, Shinshi H: Ethylene-inducible DNA-bindingproteins that interact with an ethylene-responsive element. PlantCell 1995, 7:173-182.

34. Buttner M, Singh KB: Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocselement binding protein. Proc Natl Acad Sci USA 1997,94:5961-5966.

35. Suzuki K, Suzuki N, Ohme-Takagi M, Shinshi H: Immediate earlyinduction of mRNAs for ethylene-responsive transcriptionfactors in tobacco leaf strips after cutting. Plant J 1998,15:657-665.

36. Gu YQ, Yang C, Thara VK, Zhou J, Martin GB: Pti4 is induced by •• ethylene and salicylic acid, and its product is phosphorylated by

the Pto kinase. Plant Cell 2000, 12:771-785.Wounding, exogenous ethylene and salicylic acid application, and pathogeninfection upregulate PTI4 mRNA levels. Bacterially-expressed PTI4 proteinwas shown to bind to the GCC-box in vitro, and this binding was stimulatedby co-incubation with the PTO kinase. PTO phosphorylated PTI4 on fourthreonine residues, and this phosphorylation enhanced the GCC-box-bind-ing activity of PTI4. The in vivo evidence for this phosphorylation/interactioncame from a transgenic study in which parallel overexpression of PTO andPTI4 led to increased PR-gene expression levels.

37. Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M: Arabidopsis•• ethylene-responsive element binding factors act as

transcriptional activators or repressors of GCC box-mediatedgene expression. Plant Cell 2000, 12:393-404.

Five novel Arabidopsis EREBP-related genes, AtERF1–5, were cloned andcharacterized. The AtEBP genes were differentially regulated by ethyleneand several abiotic stress factors. When expressed in E. coli, all of theAtERF proteins bound to the GCC box but not to its mutant versions in anin vitro assay. In a transient expression assay in planta, three of the five pro-teins, AtERF1, AtERF2 and AtERF5, activated the transcription, whereasAtERF3 and AtERF4 repressed the transcription, of a reporter construct thatharbored four copies of the GCC box. Combining a positive regulator (i.e.AtERF5) with a negative regulator (i.e. AtERF3) in a transient expressionassay abolished the AtEBP5-mediated transcriptional activation. Inhibition oftranscription by AtERF3 was shown to be accomplished by an active mech-anism because it does not require competition between the activator and therepressor for the same binding site on the DNA molecule.

38. Lincoln C, Britton JH, Estelle M: Growth and development of theaxr1 mutants in Arabidopsis. Plant Cell 1990, 2:1071-1080.

39. Timpte C, Lincoln C, Pickett FB, Turner J, Estelle M: The AXR1 andAUX1 genes of Arabidopsis function in separate auxin-responsepathways. Plant J 1995, 8:561-569.

40. Lehman A, Black R, Ecker JR: HOOKLESS1, an ethylene responsegene, is required for differential cell elongation in the Arabidopsishypocotyl. Cell 1996, 85:183-194.

41. Leyser HMO, Lincoln CA, Timpte C, Lammer D, Turner J, Estelle M:Arabidopsis auxin-resistance gene AXR1 encodes a proteinrelated to ubiquitin-activating enzyme-E1. Nature 1993,364:161-164.

42. Ruegger M, Dewey E, Gray WM, Hobbie L, Turner J, Estelle M: TheTIR1 protein of Arabidopsis functions in auxin response and isrelated to human SKP2 and yeast Grr1p. Gene Dev 1998,12:198-207.

43. del Pozo JC, Timpte C, Tan S, Callis J, Estelle M: The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. Science1998, 280:1760-1763.

44. Pickett FB, Wilson AK, Estelle M: The aux1 mutation of Arabidopsisconfers both auxin and ethylene resistance. Plant Physiol 1990,94:1462-1466.

45. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,Walker AR, Schulz B, Feldmann KA: Arabidopsis AUX1 gene: apermease-like regulator of root gravitropism. Science 1996,273:948-950.


46. Marchant A, Kargul J, May ST, Muller P, Delbarre A, • Perrot-Rechenmann C, Bennett MJ: AUX1 regulates root

gravitropism in Arabidopsis by facilitating auxin uptake withinroot apical tissues. EMBO J 1999, 18:2066-2073.

Significant evidence is provided to show that AUX1 is an auxin influx-carrier.First, wild-type roots take up more than twice as much labeled 2,4-D as aux1roots. Second, the expression pattern of AUX1 in the wild-type root apex isconsistent with its proposed function as the auxin influx-carrier. Third, in theroot, aux1 seedlings are insensitive to the auxins IAA and 2,4-D but not tothe diffusible auxin 1-NAA. Fourth, 1-NAA rescues the agravitropic root phe-notype of aux1 mutant, but not that of the axr2 or axr3 mutants. Fifth, theefflux-carrier inhibitor NPA blocks the ability of 1-NAA to revert the aux1agravitropic phenotype.

47. Chen RJ, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH : TheArabidopsis thaliana AGRAVITROPIC1 gene encodes acomponent of the polar-auxin-transport efflux carrier. Proc NatlAcad Sci USA 1998, 95:15112-15117.

48. Luschnig C, Gaxiola RA, Grisafi P, Fink GR: EIR1, a root-specificprotein involved in auxin transport, is required for gravitropism inArabidopsis thaliana. Gene Dev 1998, 12:2175-2187.

49. Muller A, Guan C, Gälweiler L, Tanzler P, Huijser P, Marchant A,Parry G, Bennett M, Wisman E, Palme K: AtPIN2 defines a locus ofArabidopsis for root gravitropism control. EMBO J 1998,17:6903-6911.

50. Utsuno K, Shikanai T, Yamada Y, Hashimoto T: AGR, anAGRAVITROPIC locus of Arabidopsis thaliana, encodes a novelmembrane-protein member. Plant Cell Physiol 1998,39:1111-1118.

51. Guzman P, Ecker JR: Exploiting the triple response of Arabidopsisto identify ethylene-related mutants. Plant Cell 1990, 2:513-523.

52. Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, • Watahiki MK, Yamamoto K, Liscum E: The NPH4 locus encodes the

auxin response factor ARF7, a conditional regulator of differentialgrowth in aerial Arabidopsis tissue. Plant Cell 2000, 12:757-770.

The aphototropic Arabidopsis mutant nph4 exhibits multiple auxin-relateddifferential growth defects. Ethylene gas was found to suppress thesedefects, presumably by enhancing the auxin responsiveness of themutant. Functional AXR1 and HLS1 genes, as well as normal polar trans-port of auxin, were required for the ethylene-mediated restoration of pho-totropism in the nph4 mutant. Cloning of NPH4 revealed that it encodesan auxin-response factor, ARF7, implying the existence of a redundantethylene-responsive ARF system in Arabidopsis.

53. Thomma BPHJ, Eggermont K, Tierens KFMJ, Broekaert WF: • Requirement of functional ethylene-insensitive 2 gene for efficient

resistance of Arabidopsis to infection by Botrytis cinerea. PlantPhysiol 1999, 121:1093-1101.

The ein2-1 mutant of Arabidopsis was tested for its ability to withstandinfection by three fungal pathogens. In the ein2-1 mutant, Alternaria brassi-cicola failed to trigger the systemic induction of several pathogen-relatedgenes that are highly activated upon fungal inoculation or treatment withexogenous ethylene or methyl jasmonate in wild-type plants. However,A. brassicicola caused similar non-spreading necrotic lesions in both theein2-1 mutant and wild-type plants. An incompatible interaction wasobserved in the wild-type plants and the ein2-1 mutant upon inoculation witha biotrophic fungus Peronospora parasitica. In contrast, two different strainsof the necrotrophic fungus Botrytis cinerea caused complete tissue macer-ation and death in ein2-1, but not in wild-type Arabidopsis. Pretreatment ofplants with ethylene significantly improved the resistance of the wild-typeplants, but not of ein2-1, to B. cinerea.

54. Norman-Setterblad C, Vidal S, Palva ET: Interacting signal pathways • control defense gene expression in Arabidopsis in response to

cell wall-degrading enzymes from Erwinia carotovora. Mol PlantMicrobe Interact 2000, 13:430-438.

E. carotovora infection or treatment with a culture filtrate (CF) that was richin cell-wall degrading enzymes induced local and systemic resistance inArabidopsis plants. CF-triggered gene induction was studied in wild-typeplants and in several mutants affected in ethylene, jasmonic acid (JA), or sal-icylic acid (SA) signaling/biosynthesis/accumulation. Ethylene and JA sig-naling were found to be crucial not only for CF-mediated gene induction butalso for plant resistance against the E. carotovora infection. In contrast,

although SA was able to potentiate CF induction of ethylene and JA-respon-sive genes, lack of SA did not affect the overall development of disease.Additional evidence is provided in support of multiple signaling pathwaysacting in concord.

55. Knoester M, Pieterse CMJ, Bol JF, Van Loon LC: Systemic resistance • in Arabidopsis induced by rhizobacteria requires ethylene-

dependent signaling at the site of application. Mol Plant MicrobeInteract 1999, 12:720-727.

The SAR and ISR responses of several ethylene-insensitive mutants wereexamined and compared to those of wild-type Arabidopsis plants. Whereasthe ISR elicited against Pseudomonas syringae pv. tomato (Pst) in responseto root colonization by rhizobacteria was significantly diminished in Einmutants, the SAR response induced by an avirulent strain of Pst was indis-tinguishable in wild-type and mutant plants. Studies of the eir1-1 mutantshowed that the ISR response required functional ethylene signaling at thesite of application of rhizobacteria.

56. Dong HS, Delaney TP, Bauer DW, Beer SV: Harpin induces disease • resistance in Arabidopsis through the systemic acquired

resistance pathway mediated by salicylic acid and the NIM1 gene.Plant J 1999, 20:207-215.

Induction of disease resistance by harpin, a product of the hrpN gene ofErwinia amylovora, was studied in Arabidopsis. Harpin was found toelicit a SAR response in a salicylic-acid-dependent and ethylene-/methyl-jasmonate-independent manner.

57. Ohtsubo N, Mitsuhara I, Koga M, Seo S, Ohashi Y: Ethylene • promotes the necrotic lesion formation and basic PR gene

expression in TMV-infected tobacco. Plant Cell Physiol 1999,40:808-817.

Challenging a TMV-resistant cultivar of tobacco with the virus results in ahypersensitive response, that is, in the formation of necrotic lesions that limitvirus distribution in the plant. Ethylene gas was shown to be directly involvedin restricting pathogen infection in tobacco leaves. The application of ethyl-ene biosynthesis/action inhibitors reduced lesion formation, but transgene-mediated stimulation of ethylene evolution enhanced the hypersensitiveresponse. These findings were confirmed at the molecular level.

58. Kachroo P, Yoshioka K, Shah J, Dooner HK, Klessig DF: Resistanceto turnip crinkle virus in Arabidopsis is regulated by two hostgenes and is salicylic acid dependent but NPR1, ethylene, andjasmonate independent. Plant Cell 2000, 12:677-690.

59. Ciardi JA, Tieman DM, Lund ST, Jones JB, Stall RE, Klee HJ: • Response to Xanthomonas campestris pv. vesicatoria in tomato

involves regulation of ethylene receptor gene expression. PlantPhysiol 2000, 123:81-92.

Wild-type, Nr mutant and NR-overexpressing tomato plants were infectedwith virulent and avirulent strains of X. campestris. All plants were resistantto the avirulent strain, but wild-type plants showed greater sensitivity to thevirulent pathogen than did the Nr mutant and NR-overexpressing lines. Theenhanced pathogen tolerance of the Nr mutants and NR-overexpressorswas caused by their ethylene insensitivity. In the wild-type tomato, both viru-lent and avirulent bacterial strains induced transcription of the LeETR4 andNR mRNAs, but not of the LeETR1, LeETR2 or LeETR5 mRNAs. LeETR4induction by the avirulent strain was, however, blocked in the Nr mutant. TheNr mutant also showed reduced induction of several PR genes.

60. Krasko A, Schroder HC, Perovic S, Steffen R, Kruse M, Reichert W, •• Muller IM, Muller WEG: Ethylene modulates gene expression in

cells of the marine sponge Suberites domuncula and reduces thedegree of apoptosis. J Biol Chem 1999, 274:31524-31530.

The effect of ethylene treatment on sponge metabolism was investigated.Under starvation conditions, the degree of apoptotic cell death was dimin-ished and the intracellular concentration of calcium was increased in thepresence of ethylene gas. Two ethylene responsive cDNAs, SDERR andCaM kinase II, were cloned from Suberites domuncula and their ethylene-mediated induction was analyzed. This work provides the first example ofethylene acting as a signal in an animal.

61. Beaudoin N, Serizet C, Gosti F, Giraudat J: Interactions betweenabscisic acid and ethylene signaling cascades. Plant Cell 2000,12:1103-1116.

62. Ghassemian M, Nambara E, Cutler S, Kawaide H, Kamiya Y,McCourt P: Regulation of abscisic acid signaling by the ethyleneresponse pathway in Arabidopsis. Plant Cell 2000, 12:1117-1126.


Documents

Ethylene signaling: from mutants to molecules