active oxygen species in plant signal transduction

Plant Science 161 (2001) 405414

Review

The role of active oxygen species in plant signal transduction

Frank Van Breusegem, Eva Vranova, James F. Dat 1, Dirk Inze *Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniersitair Instituut oor Biotechnologie (VIB), Uniersiteit Gent,

K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Received 31 January 2001; received in revised form 16 May 2001; accepted 16 May 2001

Abstract

Adequate responses to environmental changes are crucial for plant growth and survival. However, the molecular andbiochemical mechanisms that orchestrate these responses are still poorly understood and the signaling networks involved remainelusive. A central role for active oxygen species (AOS) during biotic and abiotic stress responses is well-recognized, although underthese situations AOS can either exacerbate damage or act as signal molecules that activate multiple defense responses. This dualitycan be obtained only when cellular levels of AOS are tightly controlled at both the production and consumption levels. Thisreview focuses on the involvement of AOS in stress signal transduction in plants, guided by a summary of work performed in ourlaboratory on plants that are deficient in catalase activity. These plants not only reveal the importance of catalase in coping withenvironmental stresses, but also provide a powerful in planta model system to study the multiple roles of hydrogen peroxide duringplant stress. 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Active oxygen species; Catalase; Defense response; Oxidative stress; Signal transduction

www.elsevier.com/locate/plantsci

1. Introduction

A wide range of environmental stresses (such as highand low temperatures, drought, salinity, UV or ozonestress and pathogen infections) is potentially harmful toplants. A common aspect of all these adverse conditionsis the enhanced production of active oxygen species(AOS) within several subcellular compartments of theplant cell [1,2]. The reduction of oxygen to waterprovides the energy necessary for the impressive com-plexity of higher organisms, but its reduction is a mixedblessing. When AOS are incompletely reduced, they canbe extremely reactive and oxidize biological molecules,such as DNA, proteins and lipids [3]. Molecular O2 isreduced through four steps, thus generating several O2radical species (Eq. (1); [4]). The reaction chain requiresinitiation at the first step, whereas subsequent steps areexothermic and can occur spontaneously, either cata-lyzed or not.

O2 (H)O2H2O2OH+H2O2H2O (1)

The first step in O2 reduction produces the relativelyshort-lived and not readily diffusable hydroperoxyl(HO2) and superoxide (O2) radicals. The half-lifefor O2 is 24 s [5,6]. These oxygen radicals arehighly reactive, forming hydroperoxides with enes anddienes [7]. Furthermore, specific amino acids, such ashistidine, methionine and tryptophan, can be oxidizedby O2 [6]. O2 will also cause lipid peroxidation in acellular environment, thereby weakening cell mem-branes [8].

Further reduction of O2 generates hydrogen peroxide(H2O2), a relatively long-lived molecule (1 ms) that candiffuse some distance from its site of production [9,10].The biological toxicity of H2O2 through oxidation ofSH groups have long been known and is enhanced bythe presence of metal catalysts through HaberWeissor Fenton-type reactions (Eq. (2)).

Fenton or HaberWeiss reactions:

O2+Fe3+Fe2++O2

H2O2+Fe2+Fe3++OH+OH

Overall: H2O2+O2OH+OH+O2 (2)

* Corresponding author. Tel.: +32-9-264-5170; fax +32-9-264-5349.

E-mail address: [email protected] (D. Inze).1 Present address: Departement de Biologie et Ecophysiologie, Uni-

versite de Franche-Comte, F-25030 Besancon Cedex, France.

0168-9452/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0168 -9452 (01 )00452 -6

F. Van Breusegem et al. / Plant Science 161 (2001) 405414406

The last species to be reduced in this reaction is thehydroxyl radical (OH). It has a very strong potentialand a half-life of 1 s. As a result, it has a very highaffinity for biological molecules at its site of produc-tion, reacting at almost diffusion-controlled rates (K109 m1 s1) [8].

AOS are mostly byproducts of the regular cellularmetabolism, but they may be generated through thedisruption of electron transport systems during stressconditions. The main sites of AOS production in theplant cell during abiotic stress are the organelles withhighly oxidizing metabolic activities or with sustainedelectron flows: chloroplasts, mitochondria and micro-bodies (reviewed in Ref. [11]). In the chloroplasts, theprimary source of H2O2 is thought to be the Mehlerreaction. Photorespiration in the peroxisomes via glyco-late oxidase is another source of H2O2 production. AOSproduction in plant mitochondria has received less at-tention in the past, but recent data suggest that theycan be a source of AOS under specific stress conditions[12,13]. During plantpathogen interactions, AOS for-mation is mechanistically similar to the oxidative burstin macrophages [14,15].

The reactive nature of AOS makes them potentiallyharmful to all cellular components. Fortunately, plantshave the capacity to cope with these reactive oxygenspecies by eliminating them with an efficient AOS-scav-enging system [16]. Because hydroxyl radicals are fartoo reactive to be controlled directly, aerobic organismsprefer to eliminate the less reactive precursor forms,superoxide and H2O2. Superoxide dismutases (SOD)are considered key players within the antioxidant de-fense system, as they regulate the cellular concentrationof O2 and H2O2 (O2

SODH2O2+O2). Various SOD

isozymes are active within the plant cell and are con-trolled developmentally and environmentally [17]. H2O2is eliminated by catalases and peroxidases. Catalasesremove the bulk of H2O2, whereas ascorbate peroxi-dases (APX) can scavenge H2O2 that is inaccessible forcatalase because of their higher affinity for H2O2 andtheir presence in different subcellular locations [18,19].Other components of the plant AOS-scavenging systeminclude all enzymes involved in the waterwater cycle[20] and low-molecular weight antioxidant molecules,such as ascorbic acid, carotenoids and glutathione [21].Under moderate stress conditions, the radicals are effi-ciently scavenged by this antioxidant defense system.However, during periods of more severe stress, thescavenging system may become saturated by the in-creased rate of radical production. Excessive levels ofAOS result in damage to the photosynthetic apparatus(photoinhibition), ultimately leading to severe cellulardamage and chlorosis of the leaves. The importance ofthe antioxidant defense system is demonstrated by thefact that overproduction of several AOS scavengers indifferent transgenic plants leads to a significant protec-

tion against oxidative stress [22]. Aside from their de-structive nature, AOS can also be used in a beneficialway by the plant. AOS play an important role ininducing protection mechanisms during both biotic andabiotic stresses. The best known example is in theactivation of resistance responses during incompatibleplantpathogen interactions. Upon infection, a plasmamembrane NADPH oxidase is activated, producingsuperoxide radicals [23] that are converted into H2O2via spontaneous dismutation or via SOD activity. Thedefensive properties of H2O2 are situated at severalstages. (i) High levels of H2O2 are toxic for bothpathogen and plant cells. Killing of the plant cellssurrounding the infection site inhibits spreading of a(biotrophic) pathogen [24]. (ii) Hydrogen peroxide canserve as a substrate in peroxidative cross-linking reac-tions of lignin precursors and induce cross-linking ofcell wall proteins. A reinforced plant cell wall slowsdown the spread of the pathogen and makes newinfections more difficult. (iii) Because H2O2 is relativelystable and diffusable through membranes (in contrastwith superoxide), it is a perfect candidate to act as asignal molecule during stress responses [2,25].

2. Catalase-deficient plants: model system for in plantaH2O2 research

The first focus on H2O2 as a potential signal in plantdefense response came with the identification of cata-lase as a salicylic acid (SA)-binding protein. Catalasewas proposed to be a receptor that becomes inactiveafter SA binding. Catalase inactivation would lead toH2O2 accumulation, which was shown to act as asecondary messenger to induce pathogenesis-related(PR) genes [26]. The specific induction of the genescoding for glutathione S-transferase (GST) and glu-tathione peroxidase (GPX) by 2 mM H2O2 in cellsuspension cultures of soybean and the ability of H2O2produced during an incompatible plantpathogen in-teraction to induce the same genes after diffusionthrough a dialysis membrane confirmed the signalingproperties of H2O2 in plants [9].

In an attempt to gather more evidence on the role ofH2O2 in plant stress signaling, our laboratory used atransgenic approach to alter H2O2 homeostasis inplanta. By using antisense and sense technology, trans-genic Nicotiana tabacum lines (CAT1AS) were pro-duced that retained only 10% of their residual catalaseactivity [27]. These transgenic plants were used to studythe local and systemic signaling role of H2O2 in patho-gen defense.

Catalases are tetrameric heme-containing enzymesthat convert 2H2O2 into O2+2H2O, primarily prevent-ing the potential damaging effects caused by changes inH2O2 homeostasis. Plants, unlike animals, have multi-

F. Van Breusegem et al. / Plant Science 161 (2001) 405414 407

ple isoforms of catalase, which are present in the perox-isomes and glyoxisomes. Catalases are the principalH2O2-scavenging enzymes in plants and can directlydismutate H2O2 or oxidize substrates, such asmethanol, ethanol, formaldehyde and formic acid. Ourlaboratory showed that plant catalases can be dividedinto three classes: class 1 catalases are most prominentin photosynthetic tissues and are involved in the re-moval of H2O2 produced during photorespiration; class2 are highly produced in vascular tissues and may playa role in lignification, their exact biological role remain-ing unknown; and class 3 are highly abundant in seedsand young plants and their activity is linked with theremoval of excessive H2O2 produced during fatty aciddegradation in the glyoxylate cycle in glyoxisomes [28].cDNAs and genes of plant catalases have been charac-terized in a wide range of species. In Arabidopsisthaliana, Nicotiana plumbaginifolia, rice and maize,cDNAs that code for the three different classes havebeen isolated [29,30]. N. plumbaginifolia contains threeactive catalase-encoding genes (Cat1, Cat2, Cat3), twoof which are expressed in mature leaves [31]. Cat1represents 80% of leaf catalase activity and is locatedin palisade parenchyma cells. Cat2 accounts for 20%of the total activity and is found mainly in the phloem.To characterize plant catalases functionally, transgenictobacco plants deficient in Cat1, Cat2 or both weregenerated in our laboratory. Transgenic tobacco lineswith strongly reduced levels of both catalase isoformswere produced by introducing a catalase-overproducingcassette. mRNA levels of both endogenous and trans-genic catalases were strongly reduced by a cosuppres-sion process. Specific catalase isozyme activities weresuppressed by using antisense cassettes of the Cat1 andCat2 genes (CAT1AS, CAT2AS). Stress assessment ofthese plants showed that catalase functions as a sink forcellular H2O2 under adverse conditions. CAT1AS to-bacco plants were more sensitive than wild-type plantsto either the redox-cycling herbicide methyl viologen orto H2O2. Enhanced sensitivity against ozone and saltstress of the CAT1AS plants demonstrated that H2O2arising from photorespiration is an important mediatorof cellular toxicity during environmental adversity andthat catalase activity is crucial for the cellular defenseagainst these stresses [32].

3. Launching the defense response

The production of these catalase-deficient transgenictobacco plants provided a unique inducible and non-in-vasive system to assess the role of changes in H2O2homeostasis in plant stress signal transduction. Underlow light (LL) conditions (100 mol/m2 per secondphotosynthetic photon fluence rate), no obvious pheno-types are observed in CAT1AS plants. Yet, exposure to

moderate or high light intensities (HL; 3001000 mol/m2 per second photosynthetic photon fluence rate) pro-duced photorespiration-dependent changes in H2O2homeostasis. The changes in H2O2 homeostasis can bemodulated depending on the intensity and duration ofthe HL exposure. Hence, the CAT1AS plants are anideal system to study the signal function of H2O2 inplanta. Perturbance of H2O2 homeostasis can be sus-tained in time and there is no need for invasive tech-niques (i.e. injections of AOS generators) to modulatethe cellular redox. But, more importantly, the use ofCAT1AS plants avoids a potential debate about thephysiological relevance of the H2O2 concentrationsused.

Whether pathogen defense-related responses could beinduced in the CAT1AS plants was first assessed (Fig.1). CAT1AS plants exposed to LL did not producebasic or acidic PR proteins constitutively. On the otherhand, in leaf tissue exposed to HL intensities, PRprotein accumulation was observed in the absence ofany pathogenic stimulus. Induction of PR proteins wasmost prominent in necrotic leaves, but could be ob-served after 2 weeks in the upper leaves without anymacroscopic damage [10]. This observation promptedus to investigate further whether localized H2O2 couldbe a signal capable of inducing local and/or systemicacquired resistance (SAR). Half parts of lower andentire upper leaves were wrapped in aluminum foil andexposed for 2 days to HL. Plants were subsequentlyreturned to LL and covered leaf parts were unwrapped.After 2 more weeks, PR-1a production was analyzed inthe covered leaf parts of CAT1AS and wild-type plants.Whereas wild-type plants did not produce PR-1a,CAT1AS plants accumulated PR-1a both in coveredparts of HL-exposed leaves and in upper leaves thathad been entirely covered [10]. This observation indi-cated that changes in H2O2 homeostasis function as atrigger for both local and systemic defense responses,similar to what is reported during pathogenesis [33,34].The in io relevance of the induced PR response inCAT1AS plants, in which defense activation was un-coupled from damage by pre-exposing them for only afew hours to HL, was confirmed by an enhanced resis-tance against the bacterial pathogen Pseudomonas sy-ringae pv. syringae [10]. Transgenic potato plants withconstitutively elevated H2O2 levels due to the overpro-duction of a fungal glucose oxidase in the apoplastshow increased acidic chitinase and anionic peroxidaselevels. The induced defense gene expression correlatedwith enhanced resistance against soft rot disease andPhytophthora infestans [35,36].

Although H2O2 is a diffusable molecule, its half-life isonly 1 ms, which essentially excludes it from being themobile signal to induce defense responses in systemictissues. This problem might be overcome by a relaymechanism. Such a model was proposed by Alvarez et


al. [34], who observed microscopic hypersensitive(HR) lesions in A. thaliana that appear throughoutthe plant upon infection with an avirulent bacterialpathogen or by injection of glucose/glucose oxidase(generating H2O2). These microbursts correlated withthe expression of defense genes (GST, PR-2) and thedevelopment of SAR. Microbursts were prevented byinjecting diphenyleneiodonium (an inhibitor ofNADPH oxidase), suggesting a central role forNADPH oxidase in this reiterative signal network.Recently, a similar relay and/or amplification mecha-nism induced by short HL changes in H2O2homeostasis was found sufficient to activate an ox-idase-dependent burst, which is required for oxidativecell death in CAT1AS plants (J. Dat, unpublishedresults).

Although the field of pathogenesis certainly led theway in oxidative stress signaling in plants for manyyears, several studies in the field of plant stress accli-mation have delivered additional evidence for a sig-naling role of H2O2 [25]. Maize seedlings injectedwith H2O2 and menadione, which is a superoxide-gen-erating compound, became more tolerant againstchilling stress. In the acclimated seedlings, chilling tol-erance was partly due to an enhanced antioxidantsystem that prevents the accumulation of AOS duringchilling stress [37,38]. Nodal potato explants subcul-tured from H2O2-treated microplants were resistant toa 15-h heat shock at 42 C (a normally lethal treat-ment) even after 4 weeks of treatment [39]. Thermo-tolerance in mustard seedlings induced by SA or heatacclimation was correlated with a transient peak inH2O2 [40]. Treatment of crowns of winter wheat with

various levels of H2O2 and a catalase inhibitor led toconcentration-dependent synthesis of proteins thatwere also induced when the plants were exposed tolow temperatures [41]. Small heat-shock proteins, in-cluding mitochondrial HSP22, accumulated in cellsuspension cultures of tomato after 2 mM H2O2 wereadded, but not in response to superoxide-generatingagents [42]. Partial exposure to excess light or injec-tion of H2O2 in Arabidopsis leaves induced protectionfrom a subsequent excess light-induced photobleach-ing [43]. This acclimation correlated with the H2O2-dependent expression of at least one antioxidant gene,the cytosolic APX. The role of H2O2 as a signal forAPX induction was further demonstrated in rice cellsuspensions. Transcript levels of cytosolic APX werealso significantly increased by H2O2 or paraquattreatment in cell suspension cultures of rice. Additionof diethyldithiocarbamate (a SOD inhibitor resultingin low H2O2 levels) reduced the induction of APX,whereas inhibition of catalase or APX (resulting inH2O2 accumulation) increased APX mRNA levels[44]. Thus, H2O2 is clearly part of the signaling cas-cade that induces cytosolic APX by providing someinformation on the signal transduction sequences andmolecular mechanisms underlying the induction of de-fense genes. We have also shown that changes inH2O2 homeostasis in CAT1AS plants induces the pro-duction of antioxidant (GPX and APX) and heat-shock proteins (HSP17.6). The induction oftheseother than PRdefense proteins was rapid(6 h) and clearly preceded the appearance of leafdamage (8 h), suggesting that induction is indepen-dent of injury (J. Dat, unpublished results). These

Fig. 1. Defense responses of CAT1AS plants. (A) Leaf of a CAT1AS plant exposed to high light (1000 mol/m2 per second photosynthetic photonfluence rate). The dark-green region was wrapped in foil during exposure. (B) Summary of responses induced during (grey) and after (black) HLexposure (courtesy of Dr Wim Van Camp).


results corroborate the role of H2O2 as a molecularsignal for the induction of gene expression that is notlimited to plantpathogen interactions. We have re-cently obtained data showing that a short and con-trolled change in H2O2 homeostasis can be exploited toimmunize plants against a subsequent stress treatment(J. Dat, unpublished results).

As already indicated by the acclimatory effects ofmenadione in maize [37], the signaling properties ofAOS are not exclusive to H2O2. In bacteria and yeast,superoxide and H2O2 induce distinct sets of defenseproteins, although the two responses overlap consider-ably [45,46]. However, increasing evidence points alsotoward differential signaling among AOS in plants.Digitonin or xanthine oxidase trigger the accumulationof a set of extensin transcripts in tomato cell suspen-sions [47]. One of these extensin transcripts is specifi-cally induced by superoxide and not by H2O2.Similarly, phytoalexin accumulation in parsley cell sus-pensions and lesion formation in lesion-stimulated dis-ease resistance in Arabidopsis mutants are specificallyinduced by superoxide and not by H2O2 [48].

4. AOS induce plant cell death

Cell death is an essential process in a plants lifecycle. Two main modes of action have been describedin plants: programmed cell death (PCD) and necrosis.PCD is controlled genetically and shares some charac-teristic features with apoptotic cell death in animalcells, such as cell shrinkage, cytoplasmic condensation,chromatin condensation and DNA fragmentation. Ne-crosis results from severe and persistent trauma and isnot considered to be orchestrated genetically [49,50].

Plant cell death has been best studied during the HR,which is typical of an incompatible plantpathogeninteraction. During the HR, an oxidative burst coin-cides with the induction of cell death at the site ofpathogen attack. This localized cell death limits thespread of the invading pathogen. The source of theoxidative burst is considered to be partly a NADPHoxidase complex and pH-dependent cell wall peroxi-dases [2,51]. However, a decrease in activity of antioxi-dant enzymes probably generates AOS during the HRof several plantpathogen interactions as well. In to-bacco cells that undergo a HR upon infiltration with afungal elicitor, accumulation of H2O2 is correlated witha decrease in CAT1 and CAT2 transcript and proteinlevels together with a decrease in total catalase activity[52]. Similarly, viral- and pathogen-induced HR-likecell death is accompanied by a post-transcriptionalsuppression of cytosolic APX production [53]. Suppres-sion of H2O2 scavenging activity probably contributesto the accumulation of threshold levels of H2O2 orchanges in H2O2 homeostasis, which are necessary forthe activation of an active cell death program.

The first direct evidence that AOS induce plant celldeath by initiating a transduction pathway rather thanby direct killing due to phytotoxic levels was providedby experiments in soybean cell cultures. A short pulseof H2O2 was sufficient to activate a hypersensitive celldeath program [9]. Five millimolar H2O2 initiated anactive cell death pathway, requiring RNA and proteinsynthesis in Arabidopsis cell suspensions. To initiate anirreversible cell death process, H2O2 had to be presentfor 60 min [54]. We have recently demonstrated that inplanta-generated changes in H2O2 homeostasis can in-duce an active cell death pathway. A transient changein H2O2 homeostasis in CAT1AS plants was sufficientto activate a PCD program similar to that observedduring the HR. In CAT1AS tobacco plants, the celldeath program that is induced by an increase in H2O2can be impeded by inhibiting de noo protein synthesis,blocking Ca2+ fluxes, kinase/phosphatase activities andan oxidase-dependent burst (J. Dat, unpublished re-sults). Thus, changes in H2O2 levels are not per se thecell death executioners, but they trigger a signal trans-duction cascade that ultimately leads to an active celldeath program. In the lesion-simulating disease resis-tance response mutant (lsd1) of Arabidopsis, superox-ide, and not H2O2, initiates a runaway cell deathphenotype, providing genetic evidence for a role ofO2 in plant cell death [48]. The lsd1 mutant grownunder long days forms spontaneously necrotic lesionson leaves and cannot stop the spreading of cell death.O2 drastically accumulates in front of the spreadingzone of cell death. Hence, O2 seems to be the criticalsignal in the cell death process, which is monitored viathe rheostat LSD1. In the ozone-sensitive rcd1 mutantof Arabidopsis, O2 is both necessary and sufficient toinitiate an ethylene-dependent cell death signaling path-way, hereby propagating cell death [55]. Thus, H2O2and O2 are indisputably involved in genetically con-trolled cell death programs in plants.

5. Decision makers during stress situations

Under environmental stress conditions, plant growthis reduced or even stopped. Upon several stress condi-tions, a decrease in cell numbers, mitotic activity or celldivision rates is observed in either leaves or roots[56,57]. The reduction of division under unfavorableconditions allows the conservation of energy, therebylaunching the appropriate defense response and alsoreducing the risk of heritable damage [58,59]. Althoughlittle is known about how various stress conditionsaffect the cell cycle, dehydration of wheat leaves re-duced the activity of the key cell cycle regulator, thecyclin-dependent kinase (CDK), probably as a result ofan inhibitory phosphorylation [60]. Oxidative stress canarrest cell division and, in animal and yeast cells,


checkpoints have been identified that lead to cell cyclearrest upon oxidative stress [61]. AOS have been shownalso to be good candidates to link stress responses andcell cycle progression in plants. Menadione impairedthe G1-to-S transition, slowed down DNA replicationand delayed the entry into mitosis in tobacco cellsuspensions [62]. Cell cycle arrest was associated withan inhibition of CDK activity, cell cycle gene expres-sion and a concomitant activation of stress genes. Sim-ilar effects were observed on tobacco plants [62].Accordingly, exogenous application of micromolar con-centrations of glutathione (GSH) increases the numberof meristematic root cells undergoing mitosis, whereasdepletion of GSH has the opposite effect [63]. A GSH-dependent developmental pathway is essential to ini-tiate and maintain cell division during post-embryonicroot development [58]. Whereas cell cycle progression isunder negative control of AOS, somatic embryogenesisis stimulated by H2O2 [64] and is essential for rootgravitropism [65]. The first molecular link that wasdemonstrated between oxidative stress and growth re-sponses is a mitogen-activated protein kinase, ANP1.H2O2 can specifically activate ANP1 that triggers aphosphorylation cascade, resulting in the induction ofspecific stress genes, but blocks simultaneously the mi-togenic action of auxin [66]. Although the exact role ofAOS during plant growth and development is stillpoorly understood, the above examples demonstratethat under environmental stress conditions, AOS canplay a decisive role at specific checkpoints during theplant cell cycle, leading to an adequate defenseresponse.

6. Part of a team: interaction between AOS and othersignaling components

It would be difficult to imagine that H2O2 is the onlysignal responsible for the orchestration of the diverseresponses described above. A close interaction withother signaling molecules and pathways is a more real-istic scenario. Ethylene, SA, jasmonic acid (JA) andnitric oxide (NO) are other well-known players in theinduction of plant defense responses against many bi-otic and abiotic stresses [6770].

The relationship between AOS and SA is well-docu-mented. SA is mainly associated with the establishmentof SAR [68]. Upon pathogen infection, levels of SAincrease in both challenged and non-challenged leavesand plants become more resistant against a subsequentinfection. In transgenic plants that do not accumulateSA because of the overexpression of the bacterial SA-dehydrogenase gene (nahG), SAR is compromised [71].Originally, H2O2 was thought to be the downstreamsignal of SA in SAR. SA and its active analogues wereproposed to bind and inactivate catalase. However,

treatment of tobacco plants with SA increased H2O2levels and H2O2 induced the production of PR-1, whichis associated with SAR [26,72]. Later studies revealedthat exogenously applied H2O2 could not induce PR-1in nahG plants [73,74] and that the induction of PRproteins in CAT1AS plants followed an SA-dependentpathway. PR induction was investigated in crosses be-tween CAT1AS and nahG plants. These plants wereimpaired for PR induction after exposure to HL. Inaddition, a biphasic (after 6 and 33 h) increase in freeSA as well as a monophasic increase in SA glucosidewas found in HL-treated CAT1AS plants [10]. Thesedata caused a debate on the location of H2O2 versus SAin the plant defense signaling pathway, because otherreports indicate that SA can enhance H2O2 accumula-tion [75,76]. A model was suggested by Van Camp et al.[77] that seems to have put a hold on this controversy.In this model, H2O2 and SA work in unison as aself-amplifying system. In support of such a model,H2O2 induces SA accumulation [10,76] and SA en-hances H2O2 accumulation [26,78]. This self-amplifica-tion loop, involving H2O2 and SA, may generatemicrobursts that intensify and spread the H2O2 signalrequired for oxidative cell death and establishment ofacquired resistance against pathogens [34,77]. Evidenceis accumulating that H2O2 and SA are also complemen-tary secondary messengers in acclimation to abioticstress; for instance, SA and H2O2 levels are enhancedduring acclimation to heat stress [40,79] and plantsbecome acclimated to heat and chilling stress by spray-ing with H2O2 or SA [39,80,81]. Thus, similar mecha-nisms that utilize both SA and H2O2 may operate toestablish immunity against environmental cues inplants.

Ethylene is another well-established signalingmolecule that has long been recognized in plant stressresponses. It is linked to stress responses followingwounding, chilling and pathogen attack [69,82]. Byusing the CAT1AS tobacco plants, an interplay withAOS was demonstrated. Enhanced production of bPR-2 in CAT1AS plants had already suggested that H2O2induces biosynthesis of stress ethylene, which was confi-rmed by a dramatic, but transient, increase in ethyleneproduction in CAT1AS plants, 23 h after exposure toHL. This result is consistent with ethylene kineticsduring pathogen infection. The ethylene peak followedaccumulation of H2O2, but preceded that of SA,thereby implying that H2O2 can work as an intermedi-ate signal upstream of both ethylene and SA duringplant stress responses [10]. Accordingly, exogenous ap-plication of H2O2 activates ethylene production in pineneedles [83]. Furthermore, ozone, which is believed toform AOS in the apoplast, induces accumulation ofethylene in tobacco plants within 1 h of the start of thetreatment. This early induction positively correlateswith ozone sensitivity. Such an early ethylene burst


does not occur in the ozone-tolerant tobacco cultivarBelB [8486]. The ethylene burst seems to originatefrom de noo ethylene synthesis, because this peak inethylene emission corresponds with elevated levels of itsprecursor, 1-aminocyclopropane-1-carboxylic acid(ACC). The ethylene burst can be blocked by inhibitorsof enzymes involved in ethylene biosynthesis enzymes,such as ACC synthase and ACC oxidase [83,87]. Re-cently, the specific activation of ethylene biosynthesisand functional ethylene signaling was shown to berequired for O2 accumulation and cell death [55].

Jasmonic acid (JA) is a stress hormone primarilyinvolved in wound stress responses. In comparison withethylene, JA has opposite effects on cell death. TheJA-insensitive and -biosynthetic Arabidopsis mutantsjar1 and fad3/7/8 show an increased magnitude of anozone-induced oxidative burst, SA accumulation andHR-like cell death [88]. Accordingly, pretreatment ofozone-sensitive A. thaliana ecotype Cvi-0 with methyljasmonate abolishes ozone-induced cell death [55]. Thisobservation is in agreement with the potentiating effectof JA on GSH synthesis, therewith limiting AOS accu-mulation and consequent cell death [89].

Nitric oxide (NO) is used by mammals to regulatevarious biological process of the immune, nervous andvascular systems [4]. It is now becoming evident thatNO is also a ubiquitous signal in plants. NO promotesleaf expansion, seed germination and de-etiolation[90,91], but it also inhibits hypocotyl and internodeelongation, induces defense genes and phytoalexin pro-duction and potentiates the induction of hypersensitivecell death [9193]. Hence, NO can provoke beneficialor harmful effects in plant cells. This dual role probablydepends on a concentration-dependent threshold win-dow and on direct interactions with AOS species [94].Because of an unpaired electron, NO readily interactswith O2 to form peroxynitrite (ONOO) that oxi-dizes DNA, lipids, protein thiols and iron clusters,resulting in impaired enzyme activities and cellulardamage. However, interaction of NO with lipid alcoxylor lipid peroxyl radicals breaks the self-perpetuatingchain reaction during lipid peroxidation [94]. The inhi-bition of some enzymes by NO may be beneficial.NO-mediated inhibition of aconitase activity may re-duce the electron flow through the mitochondrial elec-tron transport chain, thereby decreasing mitochondrialoxidative stress. Moreover, NO converts cytosolicaconitase into an Fe-regulatory protein that controlsiron homeostasis [95]. Because Fe catalyzes the Fentonreaction that produces harmful hydroxyl radicals, alimited availability of iron may prevent oxidative dam-age. Thus, NO could affect oxidative metabolism atmultiple levels either by exacerbating or counteractingAOS effects.

7. Conclusions

We have tried to summarize the wealth of data onthe role and importance of AOS during biotic andabiotic stress responses. The level and kind of AOS aredetermining factors for the type of response. H2O2 andO2 can induce different genes, in combination orseparately, thereby giving more flexibility to the AOSsignaling function. At low concentrations, AOS inducedefense genes and adaptive responses. Sublethal levelsof AOS can acclimate plants to biotic and abiotic stressconditions and reduce plant growth, probably as partof an adaptational response. Although substantial ge-nomic responses and enzyme activities are affected byAOS, the molecular mechanisms of adaptation are stillpoorly understood and the signaling pathways involvedremain elusive. At higher concentrations, AOS trigger agenetically controlled cell death program. AOS alsocommunicate with other signal molecules and pathwaysforming a network that can orchestrate downstreamresponses. The recently established role of AOS duringgrowth and morphogenesis suggests that AOS are notonly stress signal molecules, but that they are an intrin-sic signal in plant growth and development. Geneticanalysis and further physiological studies will help posi-tion AOS signals in transduction cascades and improveour understanding on how AOS are perceived andtransduced into specific downstream responses.

Acknowledgements

The authors thank Martine De Cock for help withthe manuscript and Rebecca Verbanck for the figure.This work was supported by a grant from the EuropeanUnion (BIOTECH Program ERB-BIO4-CT96-0101).FVB is indebted to the Vlaams Instituut voor de Bev-ordering van het Wetenschappelijk-Technologisch On-derzoek in de Industrie for a postdoctoral fellowshipand JFD to the European Science Foundation (ESF)for a long-term postdoctoral fellowship and to theEuropean Union for an Individual Marie CurieFellowship.

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