Reactive Oxygen Species Metabolism in Plants: Production, Detoxification and Signaling in the Stress Response

Priscilla P. Bettini1 • Elena Cosi1,2 • Daniela Bindi1 • Marcello Buiatti1*

1 Department of Evolutionary Biology “Leo Pardi”, University of Florence, Italy 2 Present address, Center for Research in Agrobiology and Pedology, Council for Research and Experimentation in Agriculture (CRA), Cascine del Riccio (FI), Italy Corresponding author : * [email protected] ABSTRACT •- • The production of reactive oxygen species (ROS), such as superoxide radical (O2 ), hydroxyl radical (OH ) and hydrogen peroxide (H2O2), in plants is a common event in metabolic and physiological processes. ROS are normally formed in photosynthesis and respiration by the chloroplast and mitochondrial electron transfer chains, respectively, and in metabolic reactions taking place in the peroxisomes. As these active oxygen species are destructive to cellular components such as lipids, nucleic acid and proteins, plant cells are equipped with non-enzymatic and enzymatic antioxidant defense systems comprising ascorbate, glutathione, phenols, catalases, superoxide dismutases and peroxidases. Biotic and abiotic stress, such as salinity stress, excess of heavy metals, mechanical shock, UV light, exposure to ozone, water deficiency and pathogen attack, also increase ROS production. In the latter case the release of ROS, referred to as the “oxidative burst”, is one of the earliest responses activated following pathogen recognition and has been suggested to play a pivotal role in the integration and the coordination of the plant defense responses. In this review we summarize the current knowledge about ROS production and oxidative defense in plants. The role of ROS will be discussed in the frame of stress responses, with emphasis on the plant-pathogen interaction. ______

Keywords: abiotic stress, oxidative burst, oxygen radicals, plant-pathogen interaction, ROS, ROS signaling Abbreviations: AsA, ascorbic acid; CAT, catalase; ET, ethylene; GSH, glutathione; JA, jasmonic acid; HR, hypersensitive response; MAPK, mitogen-activated protein kinase; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; PCD, programmed cell death; ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; SOD, superoxide dismutase

CONTENTS

INTRODUCTION...... 28 ROS GENERATION AND SCAVENGING ...... 28 ROS IN THE PLANT STRESS RESPONSE...... 31 ROS in plant-pathogen interactions...... 31 Functions of ROS in the plant defense response...... 33 ROS signal transduction ...... 34 ROS and abiotic stress: the response to ozone...... 35 CONCLUSIONS...... 36 ACKNOWLEDGEMENTS ...... 36 REFERENCES...... 36 ______

INTRODUCTION temperature extremes, excess of light, air pollutants, anoxia, heavy metals, water deficit and pathogen attack (Smirnoff The production of reactive oxygen species (ROS), also 1993; Lamb and Dixon 1997; Dat et al. 2000; Blokhina et known as reactive oxygen intermediates (ROI) or active al. 2003; Sgherri et al. 2003; Kangasjärvi et al. 2005), the oxygen species (AOS), as byproducts of metabolic pro- equilibrium between ROS generation and removal is often cesses such as respiration and photosynthesis is the price to shifted towards the former, leading to an increased produc- pay for the advantages of aerobic life. The derivatives of tion and accumulation of oxidative species. It is now clear •- molecular oxygen, including superoxide anion O2 , singlet that a common theme in the response to both biotic and abi- 1 • oxygen O2, hydroxyl radical OH and hydrogen peroxide otic stress is the generation of ROS that can be by them- H2O2 (Table 1), are in fact toxic and can damage cellular selves the primary cause of adverse effects and/or be in- constituents leading to cell death. The ability to survive volved in signal transduction pathways and changes in gene these cellular toxins depends on enzymatic and non-enzy- expression. matic detoxification mechanisms coordinately acting to re- duce cellular damage under oxidative conditions. In this ROS GENERATION AND SCAVENGING frame, Foyer and Noctor (2005) aptly described the plant cell as a network of compartments whose varying antioxi- In plant cells ROS are continuously produced as a conse- dative buffering capacities are determined by differences in quence of normal metabolism in virtually all the intracel- synthesis, transport and/or degradation of antioxidants. lular organelles, in particular in the chloroplasts, mitochon- Upon exposure to various stress conditions, such as dria and peroxisomes (Elstner 1982; Smirnoff 1993; Apel

Table 1 Mechanisms for the generation of Reactive Oxygen Species in must not be underestimated (Rhoads et al. 2006) and mito- 3 plant cells. Chl*, chlorophyll triplet state; Fdred, reduced ferrexoxin; chondrial ROS evolution can increase as a consequence of Fdox, oxidized ferredoxin; Fe-Sred, reduced Rieske FeS center; Fe-Sox, oxidative stress conditions that lead to ROS accumulation 2+ + oxidized Rieske FeS center; M, trace metals (Fe , Cu ); QA red, reduced in other cellular compartments thereby altering the normal quinone A; QA ox, oxidized quinone A; RH, lipid. mitochondrial function (Overmyer et al. 2003). Another •- Oxidative species Production mechanisms possible source of O2 in plants is the electron leakage to •- •- Superoxide radical (O2 ) Fdred + O2 Fdox + O2 oxygen during the monooxygenase reaction catalyzed by •- Fe-Sred + O2 Fe-Sox + O2 the cytochromes, in particular cytochrome P450, in the cyto- •- QA red + O2 QA ox + O2 plasm and the endoplasmic reticulum (Urban et al. 1997). + 2+ •- Cu + O2 Cu + O2 Finally, as will be discussed in more detail later, the •- •- + Hydrogen peroxide (H2O2) O2 + O2 + 2H H2O2 + O2 NADPH-dependent oxidase complex of plant plasma mem- Hydroxyl Radical (OH•) Haber-Weiss cycle: branes, pH-dependent cell wall peroxidases, germin-like •- n+ (n-1)+ O2 + M O2 + M oxalate oxidases, and amine oxidases have also been identi- (n-1)+ - • (n+1) M + H2O2 OH + OH + M fied as sources of ROS during biotic stress (Lamb and overall: Dixon 1997; Grant and Loake 2000; Mittler et al. 2004). •- • H2O2 + O2 OH + OH + O2 In order to control the accumulation of these toxic oxy- Fenton reaction: gen derivatives, in particular hydroxyl radicals and singlet 2+ 3+ • Fe +H2O2 Fe + OH + OH oxygen, plant cells are equipped with a battery of enzymatic 1 3 1 Singlet oxygen ( O2) Chl* + O2 O2 and non-enzymatic antioxidant systems to preserve the in- • •- + • Hydroperoxyl radical (HO2 ) O2 + H HO2 tegrity of proteins and other cellular components (Dat et al. • • • Lipid radical (R ) RH + OH R + H2O 2000; Apel and Hirt 2004). The main ROS-scavenging en- • • • Lipid peroxyl radical (ROO ) R + O2 ROO zymes and the reactions they catalyze are reported in Table • • ROO + RH ROOH + R 2. Plant cells possess several classes of superoxide dismu- 1 Lipid hydroperoxide (ROOH) RH + O2 ROOH tase (SOD) enzymes, considered the first line of defence against ROS (De Gara et al. 2003; Apel and Hirt 2004; Asada 2006). SODs are characterized by the presence of and Hirt 2004) (Fig. 1). In the chloroplast ROS are gene- diverse metal ions in their active site i.e. Cu-Zn, Mn and Fe rated in the photoreduction of oxygen to H2O2 by photo- and are located mainly in chloroplasts, mitochondria and system I electron transport (Mehler reaction), whose pri- peroxisomes as well as in the cytosol (del Río et al. 2002). mary product is superoxide anion, and in photodynamic re- The presence of SOD and of other enzymatic and non-enzy- actions occurring under conditions that limit electron trans- matic antioxidants has also been detected in the apoplastic fer through the photosystems, such as high light intensity space (Vanacker et al. 1998). Catalases (CAT), tetrameric and low CO2 concentration (Elstner 1982; Apel and Hirt enzymes containing a heme prosthetic group, are predomi- 2004; Mittler et al. 2004). Superoxide anion can also be nantly localized in the peroxisomes for scavenging H2O2 produced by the leaking of electrons to molecular oxygen before it can diffuse to other cellular compartments and from electron transport chains in photosystems I and II react with metal ions (Fenton reaction) to form the highly (Sgherri et al. 1996). Finally, insufficient energy dissipation reactive hydroxyl radical (Dat et al. 2000; del Río et al. during photosynthesis leads to the formation of chlorophyll 2002). Three main isoforms of catalases are known, CAT1, triplet state that can generate singlet oxygen by energy CAT2 and CAT3, present in different plant species and res- transfer to ground-state oxygen, and its production is in- ponding to various stress conditions (Dat et al. 2000). H2O2 creased during excess light stress (Apel and Hirt 2004; can also be removed very efficiently by peroxidases, that Asada 2006; Halliwell 2006). H2O2 and superoxide radical, can be either cytosolic or cell wall-bound. Ascorbate per- but also nitric oxide (NO), are produced in peroxisomes and oxidase, glutathione peroxidase and guaiacol-type peroxi- glyoxysomes (del Río et al. 2002). The generation of super- dase belong to this class (Blokhina et al. 2003). oxide involves both the xanthine oxidase reaction in the Glutathione (GSH), L-ascorbic acid (AsA), phenolic organelle matrix and a small electron transport chain at the compounds, tocopherols, carotenoids and violaxanthin, an- peroxisomal membrane level. H2O2 on the other hand theraxanthin and zeaxanthin cooperating in the xanthophyll derives from the glycolate oxidase reaction, the -oxidation cycle are the most important non-enzymatic antioxidants of fatty acids in the catabolism of lipids, the enzymatic re- (Smirnoff 1993; Gruszecki 1995; Rice-Evans et al. 1997; action of flavin oxidases and the disproportionation of Noctor and Foyer 1998; Zancani and Nagy 1999; Grace and superoxide radicals (del Río et al. 2002; Halliwell 2006). At Logan 2000; Maeda and DellaPenna 2007). In addition to variance with mammalian cells, where mitochondria are the its role in the detoxification of xenobiotics, the tripeptide main source of ROS, the contribution of the mitochondrial GSH (-Glu-Cys-Gly) is involved in the protection against electron transfer chain to ROS production in plant cells is oxidative stress by reacting chemically with ROS, such as low (Apel and Hirt 2004). This has been ascribed to the pre- H2O2, and taking part in the enzymatic ascorbate-glutathi- sence of the enzyme alternative oxidase (AOX) that limits one cycle (Halliwell-Asada-Foyer pathway) (Noctor and the production of ROS by oxidizing ubiquinol in an O2-de- Foyer 1998; Noctor et al. 2002). GSH is oxidized to gluta- pendent reaction. However, the contribution of mitochon- thione disulphide (GSSG) and regenerated by glutathione dria to ROS generation in the dark and in non-green tissues reductase that reduces GSSG to 2GSH with NADPH as co-

Table 2 Enzymatic antioxidant systems functioning in defence against oxidative injury during plant stress responses. Ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase take part in the ascorbate-glutathione cycle (Halliwell-Asada-Foyer cycle). AsA, ascorbic acid; DHA, dehydroascorbate; GSH, glutathione; GSSG, glutathione disulphide; MDHA, monodehydroascorbate; R, aliphatic, aromatic or heterocyclic group; X, sulfate, nitrite or halide group (modified from Blokhina et al. 2003). Enzymatic antioxidant EC number Reaction catalysed •- •- + Superoxide dismutase 1.15.1.1 O2 + O2 + 2H H2O2 + O2 Catalase 1.11.1.6 2H2O2 O2 + 2H2O Ascorbate peroxidase 1.11.1.11 2AsA + H2O2 2MDHA + 2H2O Monodehydroascorbate reductase 1.6.5.4 2MDHA + NAD(P)H 2AsA + NAD(P)+ Dehydroascorbate reductase 1.8.5.1 DHA + 2GSH GSSG + AsA + Glutathione reductase 1.6.4.2 NAD(P)H + GSSG NAD(P) + 2GSH Peroxidases 1.11.1.7 H2O2 + R(OH)2 2H2O + R(O)2 Violaxanthin de-epoxidase 1.10.99.3 Violaxanthin + AsA Zeaxanthin + DHA + H2O Glutathione S-transferase 2.5.1.18 RX + GSH HX + R-S-GSH

29 ROS in the plant stress response. Bettini et al.

Cell wall

Plasma SIGNAL membrane glyoxysome Endoplasmic Fatty acids LOX Membrane reticulum metabolism lipids – O2 H2O2 P450 Lipid Peroxidation

chloroplast ROS Mehler reaction

AsA-GSH cycle H2O2 SOD SOD MAPK cascades

mitochondrion Electron transport POX nucleus H2O2+2 PhOH 2 PhO +2 H2O SOD H2O2 Transcription AsA-GSH cycle AsA + 2 PhO DHA + 2PhOH Factors

H O 2 2 Gene expression Catalase Glycolate oxidase SOD vacuole Cytoplasm peroxisome SAR, PCD Apoplast Development Stress response

Fig. 1 Main sites of reactive oxygen species production in plant cells. AsA, ascorbic acid; DHA, dihydroascorbate; GSH, glutathione; LOX, lipoxygenase; MAPK, mitogen-activated protein kinase; P450, cytochrome P450; PCD, programmed cell death; PhOH, phenolic compounds; PhO, phenoxyl radicals; POX, peroxidase; ROS, reactive oxygen species; SAR, systemic acquired resistance; SOD, superoxide dismutase. factor (Table 2). There are several examples of the positive cules with antioxidant activity such as lipoic and dihydroli- correlation between GSH biosynthesis and/or accumulation poic acid (Navari-Izzo et al. 2002). The hydroperoxides 1 and enhanced stress tolerance following treatment with formed in the reaction of tocopherols with O2 can also be heavy metals, herbicide safener or in heavy metal hyperac- cleaved to tocopherylquinones in the chloroplast lumen, cumulating species (Freeman et al. 2004; Mullineaux and rendering irreversible this type of oxidative reaction Rausch 2005; Sun et al. 2005). AsA on the other hand is the (Munné-Bosch 2005; Krieger-Liszkay and Trebst 2006). As most important antioxidant in plant cells, where it exerts tocopherols are located in the membranes their primary role ROS scavenging function both directly and by the ascor- is considered to be to protect polyunsaturated fatty acid acyl bate-peroxidase reaction that reduces H2O2 to H2O (Smir- chains from oxidative damage interrupting the peroxidative noff 1993). The oxidation of ascorbate forms the radical chain reaction (Munné-Bosch and Alegre 2002; Maeda and monodehydroascorbate (MDHA) that can be either reduced DellaPenna 2007). However, recent data seem to sustain a to ascorbate by ferredoxin, cytochrome b561 or reductases, reshaping of the role of tocopherols in the protection against or dismutated to dehydroascorbate (DHA). Dehydroascor- oxidative stress imposed by high light by virtue of the sur- bate in turn can be reduced by specific reductases (DHARs), prisingly poor phenotypic effects observed in tocopherol- glutathione-S-transferases, GSH, thioredoxins and glutare- deficient mutants and leading to propose that they are but doxins (Noctor 2006). For instance, sensitivity to the air one of numerous components taking part in this function pollutant ozone is correlated with the ascorbate status of the (Maeda and DellaPenna 2007). Another class of nonenzy- leaf tissue (Kangasjärvi et al. 2005) and AsA deficiency in matic scavengers is represented by phenolic compounds, the Arabidopsis thaliana mutant vitamin c-1 (vtc1) deter- the most abundant class of plant secondary metabolites mines enhanced sensitivity to oxidative stress caused by ex- derived from the phenylpropanoid biosynthetic pathway, in- posure to ozone, sulphur dioxide, low temperatures and UV- cluding anthocyanins, flavonoids and isoflavonoids, tannins, B light (Conklin et al. 1996). coumarins, stilbenes and structural polymers such as lignin Photosynthetic organisms, i.e. plants, algae and most and suberin (Dixon and Paiva 1995; Grace and Logan 2000). cyanobacteria, also synthesize the lipophilic antioxidants , The antioxidant capacity of polyphenols derives from their , and -tocopherols (vitamin E) (Krieger-Liszkay and properties as reducing agents as well as from their ability Trebst 2006; Maeda and DellaPenna 2007). In particular, - for metal chelation that inhibits the formation of hydroxyl tocopherol has the capacity of quenching singlet oxygen radical by the Fenton reaction (Rice-Evans et al. 1997; (Munné-Bosch 2005), while -tocopherol can react with NO Grace and Logan 2000). The reduced state of phenolics, ne- forming 5-nitro--tocopherol, therefore modulating NO en- cessary for their effective function as antioxidants, is main- dogenous level in plant tissues (Desel et al. 2007). The oxi- tained by the ascorbate present in the vacuole that can also dized tocopherol radicals and hydroperoxides thus formed function as a chemical reductant of both quinones and semi- are recycled by reductants such as ascorbate or GSH (Krie- quinones (Yamasaki and Grace 1998). The biosynthesis of ger-Liszkay and Trebst 2006). GSSG and DHA are then re- phenols is induced upon exposure to environmental stresses generated in a non-enzymatic pathway involving other mole- such as high light, UV-B, metals, wounding/herbivory or

30 Plant Stress 2 (1), 28-39 ©2008 Global Science Books pathogen attack (Dixon and Paiva 1995; Grace and Logan non-specific and specific responses must be distinguished. 2000; Winkel-Shirley 2002). Sgherri et al. (2003) by study- The non-specific responses are displayed by every plant ing the effect of copper excess on Raphanus sativus showed towards most potentially pathogenic microbes and are a that the content of phenolic acids, such as chlorogenic, caf- major component of the so called non-host resistance feic and p-coumaric acid as well as of total and reduced as- (Heath 2000). The specific responses on the other hand are corbate, increased with the intensification of Cu treatment triggered by the interaction between plant genotypes pos- and proposed an interrelation between the cytoplasmic sessing specific resistance (R) genes and pathogen geno- ascorbate-gluthatione cycle and the phenols stored in the types possessing the corresponding avirulence (Avr) genes vacuole, according to the results of Takahama and Oniki (incompatible interaction). This gene-for-gene mechanism (1997) and Zancani and Nagy (1999). (Flor 1947) leads to the induction of a rapid defense res- The regulation of the intracellular concentration of ROS ponse, the hypersensitive response (HR), characterized by is based on the equilibrium between their production and localized cell death at the site of infection (Dangl and Jones removal, the so-called called “redox homeostasis” (Mittler 2001), and to the acquirement of long-lasting, broad-spec- 2002; Foyer and Noctor 2005). The antioxidant system has trum Systemic Acquired Resistance (SAR) to subsequent therefore a dual function, (i) to modulate the concentration infections induced in plants surviving pathogen challenge of ROS and set the threshold for the induction of the ap- (Ryals et al. 1994). When the plant is susceptible to the in- propriate responses to different stress conditions and (ii) to fection by a virulent pathogen the interaction is called com- scavenge excess ROS before they can damage cellular con- patible and disease ensues. The earliest events in the resis- stituents. The efficient removal of ROS at their generation tance response are calcium influx, alkalinization of the sites depends on the sub-cellular localization of the antioxi- extracellular space, production of ROS (the “oxidative dant mechanisms, which is particularly true for the photo- burst”) and NO, and protein kinase activation (Dangl and synthetic apparatus as the main site for ROS formation in Jones 2001). The onset of the HR is also accompanied by a plant cells and the target of abiotic stressors such as high general “transcriptional reprogramming” induced by patho- light, sulphur dioxide and photodynamic herbicides (Al- gen challenge that involves the coordinated expression of scher et al. 1997). Furthermore enzymatic and non-enzyma- numerous genes coding for transcription factors, pathoge- tic scavengers, in addition to their individual capacity for nesis-related or PR-proteins, enzymes involved in bioche- ROS detoxification, take part in complex cycles of redox mical pathways from primary and secondary metabolism reactions (the Halliwell-Asada-Foyer cycle, the ascorbate and leading to the production of phytoalexins, lignin and peroxidase cycle, the water-water cycle) (Elstner 1982; salicylic acid (SA) (Hahlbrock et al. 2003). Mittler 2002; Asada 2006) where they cooperate in scaven- The first evidence for an oxidative burst in the plant ging ROS and regenerating the active forms of antioxidants response to pathogens was reported by N. Doke (1983a) in to keep the pool fully functional. The importance of the potato tuber tissue infected with the late blight oomycete antioxidant network can be further inferred from its redun- Phytophthora infestans or treated with cell wall components dancy whose aim is to counterbalance possible failures in (CWC) from the same pathogen. The production of super- one or more elements. Knockout and antisense lines for key oxide anion was observed only in response to an incom- enzymes in the pathway such as CAT, ascorbate peroxidase, patible, but not to a compatible, race of the pathogen and peroxiredoxin and SOD are viable and show compensatory was inhibited by the addition of SOD. In potato protoplasts increases in the expression of other genes with similar func- treated with the same elicitor superoxide generation could tion (Mittler et al. 2004); in mutants with low AsA con- be detected as fast as 2 min after the addition of CWC to the centration, the tocopherol pool is increased; finally, high reaction mixture, making this the earliest pathogen response light sensitivity in Arabidopsis can be obtained only in dou- known (Doke 1983b). Using a fluorescence transition assay ble mutants defective in both GSH and tocopherol (Krieger- as detection method, Apostol et al. (1989) showed in soy- Liszkay and Trebst 2006). bean cell suspension cultures that the treatment with an elicitor from the fungus Verticillium dahliae stimulated an oxi- ROS IN THE PLANT STRESS RESPONSE dative burst detectable less than 1 min after elicitor addition. This burst was mainly derived from the rapid formation of Plants are sessile organisms and as such are particularly H2O2 and its use by extracellular peroxidases to oxidize sus- susceptible to environmental fluctuations. The key for their ceptible substrates. Since these pioneering studies the in- survival is the ability to sense these changes and for this volvement of ROS in the interaction between plants and a reason they have evolved highly effective and coordinated large number of viral, bacterial and fungal pathogens as mechanisms to perceive and respond to stress. To the same well as upon treatment with various elicitors of the defense end, plants are endowed with a remarkable amount of phe- response has been described (Keppler et al. 1989; Vera- notypic plasticity, i.e. the capacity to modify their develop- Estrella et al. 1992; May et al. 1996; Wojtaszek 1997 and ment, physiology and life history according to the environ- references therein). Further experimental evidence of ROS mental conditions (Sultan 2000). Adaptive plastic responses involvement in the defense response came from experi- include the constraints imposed on growth and development ments aimed at obtaining pathogen-resistant transgenic by environmental conditions and adaptive changes that gua- plants through the manipulation of ROS metabolism. In- rantee that a given organism will maintain its function and creased H2O2 production in potato due to the insertion of a reproduce in multiple environments. They affect not only glucose oxidase enzyme from Aspergillus niger conferred the contingent success of organisms in their natural context, to plants a broad-spectrum disease resistance resulting from but also their ability for colonization of different ecological induction of specific genes, SA accumulation and cell wall niches and for evolutionary diversification. lignification (Wu et al. 1995). As previously mentioned, the production and accumula- The production of ROS follows a different time course tion of reactive oxygen species is a common feature in plant in the incompatible with respect to the compatible inter- stress (Dat et al. 2000; Baier et al. 2005; Torres et al. 2006). action. In the former the burst is biphasic, characterized by Besides their role in the onset and modulation of the biotic a transient first phase with low amplitude, considered to be and abiotic stress response, ROS participate in a range of a non specific reaction, followed by a secondary, more sus- biological processes such as senescence, phytohormone sig- tained phase of higher magnitude (Lamb and Dixon 1997). naling, stomatal closure, cell expansion and plant develop- In the presence of virulent pathogens only the first phase is ment (Laloi et al. 2004; Bhattacharjee 2005; Torres and induced. A biphasic oxidative burst is also observed in the Dangl 2005) (Fig. 1). response to ozone (Kangasjärvi et al. 2005) and in the presence of excess of metals such as copper (Raeymaekers et ROS in plant-pathogen interactions al. 2003). One of the most striking features of the oxidative burst When plant defenses to pathogens are taken into account observed in plant cells undergoing pathogen attack is its

31 ROS in the plant stress response. Bettini et al. similarity to the innate immune response in higher animals, Botrytis cinerea is derived from the activity of peroxidase where the production of superoxide by granulocytes and its and superoxide dismutase in the apoplast (Patykowski and subsequent dismutation to hydrogen peroxide have been Urbanek 2003). shown to have a role in the phagocytosis process. ROS It is therefore clear that different enzymes can be the seem not to have a direct function in killing the invading source of ROS for the oxidative burst. However, understan- microorganisms, as was originally thought, but rather to ding the contribution and the interactions of the diverse determine a pH change in the phagocytic vacuole in order ROS-generating systems in the plant defense response is to create an optimal environment for the action of enzymes not an easy task. In order to explain the role of peroxidases such as neutral proteases (Segal 2005). In mammalian cells and NADPH oxidases in the oxidative burst and in defense, a NADPH oxidase called the respiratory burst NADPH Bindschedler et al. (2006) have proposed a model where oxidase (RBO) is responsible for ROS generation (Babior et apoplastic peroxidases generate a first burst of ROS upon al. 2002). The mammalian enzyme is constituted by five recognition of pathogen-associated molecular patterns subunits, the two plasma membrane proteins gp91phox and (PAMPs) associated with basal resistance. Reactive oxygen p22phox, that occur as a heterodimeric flavohemoprotein species thus produced could subsequently activate NADPH known as cytochrome b558, and a cytosolic complex of three oxidases for the generation of the plasma membrane-associ- regulatory proteins p40phox, p47phox, p67phox. Upon stimula- ated oxidative burst in incompatible interactions. tion the regulatory complex moves to the plasma membrane The generation and accumulation of ROS in the course and associates with the cytochrome b558 to form the active of the plant defense response is accompanied by changes in oxidase. For the activation of the complex the low mole- the cellular antioxidative systems. The activities of ROS- cular weight GTPase(s) Rac1 or Rac2 are also required. As scavenging enzymes and antioxidants have been found to the production of ROS in plants can be blocked by specific decrease or increase depending on the plant-pathogen sys- inhibitors of the mammalian NADPH oxidase such as di- tem studied making it difficult to define a clear and univo- phenylene iodonium (DPI), plant homologues of the animal cal role for these systems. On one hand it is clear that ROS genes have been searched for leading to the isolation of accumulation can be achieved either by an enhanced pro- several homologues of gp91phox (rboh genes) in A. thaliana, duction and/or a lower removal, so a decrease in antioxi- rice, tomato and potato (Torres and Dangl 2005). The plant dants or scavenging enzymes activity could be necessary to Rboh proteins are larger than their mammalian counterparts. ROS build up during the defense response for pathogen kil- The C-terminal domain shows significant homology and a ling and signaling purposes. Antioxidant enzymes activity conserved membrane topology to the human gp91phox while was found to decrease in plant-virus interactions, where the the extended hydrophilic N-terminal domain has two EF resulting oxidative stress could contribute to virus replica- hand motifs that bind Ca2+ ions and a significant similarity tion and spreading (Clarke et al. 2002; Hernández et al. to the human RanGTPase-activating protein 1 (RanGAP1), 2004), as well as in tomato roots reacting hypersensitively a key regulator of the Ras-related nuclear small GTP- to the infection by the nematode Meloidogyne incognita, binding protein Ran (Keller et al. 1998). This finding also concomitant with an increase in ROS generation leading to suggests a different regulatory mechanism for the activation accumulation of ROS and cell death (Zacheo and Bleve- of the oxidative burst in plants. The N-terminal domain Zacheo 1988). A diminished activity of class I and II CAT could project into the cytoplasm and respond to the increase and of ascorbate peroxidase has also been linked to the on- in Ca2+ induced by pathogen avirulence factors, leading to set of hypersensitive cell death in the interaction between the rapid induction of the oxidative burst to initiate the HR tobacco and tobacco mosaic virus (TMV) (Dorey et al. in infected cells near to the pathogen penetration site 1998; Mittler et al. 1998). On the other hand, in incompati- (Keller et al. 1998). The phosphorylation of specific serine ble interactions an increase in ROS scavengers has been residues in the N-terminal region of the NADPH oxidase by observed as well and has been related to the need to control a calcium-dependent protein kinase (CDPK) has recently ROS production before they can damage extensively the been proposed by Kobayashi et al. (2007) to take part in the tissue. An increase in the activity of class II CAT, glutathi- regulation of Rboh in potato. Furthermore in rice, as in one reductase and peroxidase was detected following virus animal cells, a homologue of the human Rac regulatory infection or elicitor treatment in tobacco (Montalbini and subunit of the NADPH oxidase complex (OsRac1) has been Buonaurio 1995; Dorey et al. 1998) and cowpea (Moshati shown to regulate ROS production by NADPH oxidase and et al. 1993), where it was involved in the cross-linking of cell death (Kawasaki et al. 1999), and to be involved in dis- cell wall components. By comparing different species of the ease resistance acting downstream of the R genes (Oono et genus Lactuca resistant or susceptible to the pathogen Bre- al. 2001). In A. thaliana the rboh genes constitute a family mia lactucae Sedlarova et al. (2007) found that only the of ten members (AtrbohA to J) (Torres and Dangl 2005). resistant genotypes were characterized by accumulation of The isolation of Atrboh mutants allowed the identification H2O2 in infected cells together with enhanced activity of the of two genes, AtrbohD and F, specifically involved in the corresponding scavenging enzymes. Similar results have accumulation of ROS during the resistance response to bac- been obtained in the wheat (Triticum aestivum)-Fusarium terial and fungal pathogens (Torres et al. 2002). In parti- graminearum interaction, where an up-regulation of anti- cular, AtrbohD, and its tobacco counterpart NtrbohD, are oxidants was detected only in the resistant cultivar ‘Ning responsible for ROS production during the interaction with 7840’ (Zhou et al. 2005). However, Montalbini and Buo- avirulent pathogens or after elicitor treatment, while the naurio (1986) and Buonaurio et al. (1987) observed that the contribution of AtrbohF is more limited and seems to be increased SOD activity in both the Nicotiana - TMV and important in the regulation of HR. These genes probably en- Phaseolus vulgaris - Uromyces phaseoli interactions was code components of a plant NADPH oxidase that produce not sufficient to protect tissues from oxygen toxicity in the superoxide. course of the HR. An up-regulation of ROS-scavenging en- Besides the plasma membrane-bound NADPH oxidase zymes in the course of the interaction between plants and complex, the accumulation of ROS can also be due to apo- pathogens has also been detected in transcriptome analyses plastic enzymes such as peroxidases, amine, diamine and by means of the DNA microarray technology. In a compre- polyamine oxidases, and oxalate oxidases (Grant and Loake hensive study of A. thaliana transcriptome changes during 2000; Mittler et al. 2004). For instance, a germin-like oxa- SAR, Maleck et al. (2000) found that the expression of late oxidase protein that can produce H2O2 from oxalic acid genes encoding proteins involved in redox regulation such has been identified in the incompatible interaction between as peroxidase C and glutathione-S-transferase (GST) was barley and powdery mildew (Blumeria graminis f. sp. hor- upregulated. The expression levels of genes involved in free dei) (Zhang et al. 1995), and the involvement of an apo- radical scavenging were also increased in A. thaliana after plastic peroxidase in the oxidative burst has recently been inoculation with an incompatible strain of the fungus Alter- reported in A. thaliana (Bindschedler et al. 2006). Finally, naria brassicicola (Schenk et al. 2000) and in a resistant H2O2 produced in tomato leaves infected with the fungus rice cultivar upon infection with the leaf blight pathogen

Xanthomonas oryzae pv. oryzae (Kottapalli et al. 2007). 2005). In the tocopherol-deficient Arabidopsis mutant vita- It must also be mentioned that, in the course of the host- min e2 (vte2) (Sattler et al. 2006) and following infection pathogen coevolution race, some pathogens have acquired with B. cinerea in tomato or induction of peroxide stress in the ability to manipulate the ROS-scavenging system and tobacco suspension cells (Thoma et al. 2003), the increase exploit ROS production to their own advantage. This beha- in general oxidation and non-enzymatic lipid peroxidation viour is typically observed in the infections by necrotrophic products (malondialdehyde and phytoprostanes, respec- pathogens, such as B. cinerea, where a decreased level of tively) induced the accumulation of phytoalexins and the antioxidants facilitates infection by promoting the spreading expression of genes involved in signal transduction, pri- of cell death (de Gara et al. 2003; Torres et al. 2006). mary and secondary metabolism. Once more, a common theme in the defensive responses of plants and animals can Functions of ROS in the plant defense response be detected, as phytoprostanes belong to the chemical class of isoprostanes, mediators of oxidative stress in animals ROS produced in the oxidative burst following pathogen produced from arachidonic acid by a free-radical catalyzed attack or elicitor treatment have multiple roles in the biotic mechanism (Roberts and Morrow 1997). stress response of plants that are summarized in Fig. 2. During the plant defense response H2O2 accumulation The peroxidation of membrane lipids is a commonly from the oxidative burst at the cell surface can drive the observed event in the HR, where the resulting oxidative oxidative cross-linking of cell wall structural proteins, i.e. membrane damage may lead to cell death (Croft et al. 1990; Hydroxyproline-Rich Glycoproteins (HRGPs) and tyrosine- May et al. 1996). Lipid peroxidation can occur both enzy- rich proteins, after pathogen attack or elicitation contribu- matically and non-enzymatically, by the action of lipoxyge- ting to the toughening of the cell wall barrier to pathogen nases (LOX) or of reactive oxygen species, respectively. ingress (Lamb and Dixon 1997; Wojtaszek 1997; Dat et al. LOX enzymes catalyze the hydroperoxidation of fatty acids 2000). Both the insolubilization of HRGPs in the cell wall and lead to the production of active oxygen species such as (Bradley et al. 1992) and the inhibition of protoplast release singlet oxygen, superoxide radicals and hydroxyl radicals by cell-wall degrading enzymes (Brisson et al. 1994) have derived from the latter by the action of SOD (Slusarenko et been observed after treatment of bean or soybean cells with al. 1991). The resulting fatty acid hydroperoxides degene- glucan elicitor from the oomycete Phytophthora mega- rate autocatalytically producing radicals that can propagate sperma f. sp. glycinea or H2O2. The response showed the lipid peroxidation as a chain reaction (Fig. 3). Peroxidized same time course as the oxidative burst, and was inhibited fatty acids metabolites have been implicated as signal mole- by the addition of antioxidants such as catalase or AsA. cules in the activation of plant defenses as the production of ROS are also involved in cell death, a prominent feature the phytoalexin glyceollin in soybean (Degousée et al. of the HR in incompatible plant-pathogen interactions 1994), or in cell death (Knight et al. 2001; Montillet et al. where it is linked to disease resistance and has been proposed to block the spreading of the pathogen (Greenberg and Yao 2004; Van Breusegem and Dat 2006). The death of hypersensitively reacting cells, however, is not a passive Pathogen, Elicitor, PAMP necrotic process, but an active form of programmed cell death (PCD). Furthermore, in order to avoid that the spread- Ca2+ Recognition ing of cell death would irreversibly damage the whole tissue, NADPH oxidase complex the PCD programme is strictly regulated. The genes involved in PCD pathways have been identified through the CDPK isolation of lesion mimic mutants, such as A. thaliana lsd1 (lesion-simulating disease resistance response 1) where the HR is not properly controlled and cell death spreads through- PLD ROS ROS out the infected leaf (Lorrain et al. 2003). scavenging In cells undergoing hypersensitive death the main events are vacuole collapse, ion fluxes (K+, Cl- efflux, Ca2+ influx), changes in pH and depolarization of the plasma PA membrane together with chromatin condensation, DNA laddering and cytochrome c (Cyt c) release (Jones 2001; Hofius et al. 2007). The latter features correspond to well- known markers of apoptosis in animal cells, where several Direct Lipid Cell wall Defense genes Cell death SAR stimuli activate a self-destruction programme requiring Cyt toxicity peroxidation toughening activation c release for the induction of a cysteine protease (caspase) cascade (Hengartner 2000). The proteolytic attack of cas- Fig. 2 Production and functions of reactive oxygen species in the plant pases on different substrates causes the typical features of defense response. CDPK, calcium-dependent protein kinase; PA, phos- PCD such as nuclear shrinking and budding, loss of cell phatidic acid; PLD, phospholipase D; PAMP, Pathogen Associated Mole- shape, DNA laddering and ultimately leads to cell death. cular Pattern; ROS, reactive oxygen species; SAR, Systemic Acquired Support to this similarity came from the finding that a cas- Resistance. pase-like proteinase activity is involved in hypersensitive cell death in the interaction between tobacco and an incompatible strain of Pseudomonas syringae pv. phaseolicola (Del Pozo and Lam 1998). However, no homologues of RH animal caspases have been identified in plant genomes to date, but only putative caspase-like families called metacas- ROOH pases (Watanabe and Lam 2004; Hofius et al. 2007). ROOᔊ The involvement of ROS in hypersensitive cell death + Rᔊ has been demonstrated both pharmacologically, by showing that its typical features in plant cells could be blocked by ᔊ treatment with antioxidants, inhibitors of transcription and R + O2 translation (indicating the requirement for de novo protein synthesis) or of components of the signal transduction pathways, and genetically by the isolation of lesion mimic mu- Fig. 3 Chain reaction for the propagation of lipid peroxidation among tants and by the characterization of transgenic plants with the plasmamembrane fatty acids. R, lipid radical; RH, lipid; ROO, altered antioxidant levels (Lorrain et al. 2003; Van Breuse- lipid peroxy radical; ROOH, lipid hydroperoxide. gem and Dat 2006). Being very reactive, some ROS like

33 ROS in the plant stress response. Bettini et al. hydroxyl radicals or singlet oxygen have obviously the pot- transduction chain leading to the HR and to the activation ential to cause cell death by directly damaging cellular con- of the defense response, and in fact its inhibition blocks stituents. However, the link between ROS and cell death is ROS production (Jabs et al. 1997 and references therein). not as straightforward as it could be thought at first sight. The activity of calcium-permeable ion channels is modu- The oxidative burst per se is not sufficient to trigger cell lated by cyclic nucleotides and by heterotrimeric G protein- death and its interaction with NO and phytohormones is dependent phosphorylation (Lecourieux et al. 2006; Ma and necessary to this end. Concerning NO, a signal molecule in Berkowitz 2007). Furthermore, calcium can exert a direct the vertebrate vascular, nervous and immune systems, control on ROS generation at the level of the NADPH oxi- Delledonne et al. (2001) have shown that in plant, as in dase complex by binding to the EF hands in the N-terminal animal, cells its cooperation with ROS is necessary for the domain of plant gp91phox homologues (Keller et al. 1998; induction of the hypersensitive reaction and that ROS and Sagi and Fluhr 2001) and via the phosphorylation of ser re- NO production must be fine-tuned to efficiently induce the sidues in the same domain by a calcium-dependent protein •- 2+ HR. In animal macrophages NO and O2 react to form kinase (Kobayashi et al. 2007). As the transduction of Ca peroxynitrite (ONOO-), which is toxic to pathogen and signals is based on calmodulin, a small acidic protein pre- tumor cells. On the contrary in plant cells ONOO- is not an sent in all eukaryotes that binds Ca2+ and activates down- essential intermediate for hypersensitive cell death and stream targets, its involvement in the plant oxidative burst there is no evidence for a direct interaction between NO and has been investigated. In this frame, the enzymes NAD •- O2 . Induction of cell death and killing of the pathogen are kinase and catalase have been identified as targets for cal- achieved instead by the synergistic interaction between NO modulin action (Harding et al. 1997; Yang and Poovaiah and H2O2 produced from superoxide by SOD during the HR. 2002), pointing to a dual role of calcium in the regulation of However, NO can have a dual role in the modulation of H2O2 homeostasis: a positive regulation that could act plant cell death: it cooperates with ROS in its induction and directly through the activation of NADPH oxidase or indi- can also protect plant cells from ROS damage by increasing rectly on NAD kinase, and a negative regulation through the levels of scavenging enzymes such as CAT, SOD, GST the stimulation of catalase. Recent data have demonstrated and AOX (Wendehenne et al. 2004). that signal transduction downstream from Ca2+ influx and The phytohormones SA, ethylene (ET) and jasmonic leading to the HR also requires NO, whose biosynthetic en- acid (JA) are also implicated in the control of cell death zyme nitric oxide synthase can be activated through calmo- induced by ROS (Hofius et al. 2007). The effect of SA dulin by an increase of Ca2+ in the cytoplasm (Ma and Ber- seems to depend on a concentration gradient at the infection kowitz 2007). NADPH oxidase activity and ROS genera- site. Where SA concentration is high, i. e. at the very site of tion are modulated also by lipid-based signal transduction pathogen penetration, cell death is promoted to block systems i.e. phospholipase D (PLD) and its product phos- pathogen growth and diffusion, while low levels of SA in phatidic acid (PA) (Sang et al. 2001). PLD gene expression the adjacent cells promote cell survival and impede the and PA production have been observed following exposure spreading of the lesions. The spreading of cell death from to H2O2, elicitors and pathogens, and induce the expression HR sites is controlled also by ROS produced by the of elicitor-responsive genes and the biosynthesis of phyto- respiratory burst NADPH oxidase homologues AtrbohD alexins (Wang et al. 2006). Furthermore, PA produced by and AtrbohF, which negatively regulate cell death induced phospholipases D and C can function as a second mes- by SA or its analog benzothiadiazol (Torres et al. 2005). senger activating protein kinase cascades, CDPK and affec- McDowell and Dangl (2000) have proposed that SA, NO ting ion channels activity (Laxalt and Munnik 2002). and ROS could act together in the modulation of cell death. The signaling pathway downstream from ROS includes The production of ROS and NO upon recognition of an mitogen-activated protein kinases (MAPK) cascades typ- avirulent pathogen stimulates the biosynthesis of SA as both ically represented by three protein kinases, MAPKKK, NO and ROS can up-regulate the transcription of Phe am- MAPKK and MAPK (Asai et al. 2002; Nakagami et al. monia lyase, a key enzyme in phenylpropanoid biosynthesis 2005). Mitogen-activated protein kinases responding to (Hancock et al. 2002). SA in turn enhances the responses pathogens and elicitor molecules have been identified in a induced by ROS and NO creating a cycle of positive feed- number of plants, including A. thaliana, tobacco, parsley, back regulation that amplifies the initial signal. Salicylic alfalfa, rice and tomato (Nakagami et al. 2005). The to- acid interacts also with ET in the regulation of PCD, cre- bacco MEK2, salicylate-induced (SIPK) and wound-in- ating a feedback cycle that positively influences the ROS- duced protein kinases (WIPK) and their Arabidopsis ortho- dependent cell death. However, in some cases a PCD path- logues MPK3 and MPK6, are also induced by H2O2. In way independent of ET has been identified (Van Breusegem particular, the activation of MEK3 and MEK6 in response and Dat 2006; Hofius et al. 2007). Finally, JA seems to con- to H2O2 is mediated by the MAPKKK ANP1 and by a trol lesion formation, even if this event can be JA-inde- nucleoside diphosphate kinase 2 (AtNDPK2) (Nakagami et pendent as well (Lorrain et al. 2003; Greenberg and Yao al. 2005; Fujita et al. 2006). The A. thaliana OXI1 (OXI- 2004). The effect of JA in the modulation of the cell death DATIVE SIGNAL INDUCIBLE1) putative serine/threonine response depends on the chemical nature of the oxygen ra- kinase has been recently identified as a further signaling dical signal (superoxide/H2O2 versus singlet oxygen) and on element upstream from the MAPK cascade (Rentel et al. its concentration with respect to other phytohormones such 2004). The final targets of these MAPK cascades are trans- as SA (Van Breusegem and Dat 2006; Hofius et al. 2007). cription factors that modulate the expression of specific genes in a stimulus-dependent manner, and in fact ROS ROS signal transduction increase the expression of members of several families of transcription factors such as WRKY and Myb (Laloi et al. Both superoxide anion and H2O2 are involved in signal 2004; Mittler et al. 2004). The activation of gene expression transduction pathways leading to the plant defense response by transcription factors can also be controlled by the redox at the local and the systemic level. There is evidence that balance of the cell as has been shown for NPR1, an essen- calcium ions and calcium-binding proteins, lipid-based sig- tial mediator of SAR (Dong 2004). Upon the SA-induced nals, protein kinase cascades and G proteins are involved in onset of SAR, NPR1 is localized to the nucleus (Kinkema et these pathways (McDowell and Dangl 2000; Nakagami et al. 2000) where it interacts with basic leucine zipper type al. 2005; Lecourieux et al. 2006; Torres et al. 2006; Laxalt (bZIP) transcription factors belonging to the TGA family and Munnik 2002; Ma and Berkowitz 2007) (Figs. 1, 2). for the activation of the expression of genes encoding PR- A modification of ion fluxes, and in particular an in- proteins (Zhang et al. 1999). In non-induced conditions crease in cytosolic Ca2+ concentration, is one of the earliest NPR1 is present as a large, inactive oligomeric complex events induced by pathogen attack or elicitor treatment. An whose monomers are held together by disulphide bonds. influx of Ca2+ ions through the plasma membrane ion chan- The oxidative burst that occurs upon SAR induction estab- nels is considered an essential component in the signal lishes a transient increase in the cellular reduction potential

34 Plant Stress 2 (1), 28-39 ©2008 Global Science Books leading to a conformational change in the NPR1 complex HR, is correlated with the rapid accumulation of SA and and to the appearance of active monomers that localize in requires a SA-dependent signaling pathway (Sharma and the nucleus and activate PR gene expression (Mou et al. Davis 1994; Sharma et al. 1996; Sandermann et al. 1998). 2003). By examining in detail the interaction between Genes whose expression is increased upon exposure to NPR1 and the transcription factors TGA1 and TGA4, Des- ozone include those involved in the flavonoid and phenyl- prés et al. (2003) have shown that in non-induced condi- propanoid pathways, SA-signaling, ethylene and JA biosyn- tions both transcription factors are kept in an inactive state thesis as well as encoding PR proteins and antioxidant en- through the formation of an intramolecular disulphide bond zymes aimed at decreasing the level of toxic intracellular between two oxidized cysteine residues. The induction of ROS (Sharma and Davis 1994; Li et al. 2006; Tosti et al. SAR causes the reduction of the disulphide bridges and the 2006). Consistent with the known reduction in photosyn- breakage of the bonds thus allowing the interaction NPR1/ thesis and induction of senescence, ribulose-1,5-bisphos- TGA and the subsequent activation of PR gene expression. phate carboxylase/oxygenase large and small subunit and How the reduced cellular state is established as a conse- binding protein genes were down-regulated, while senes- quence of the SA-triggered initial oxidative burst is not cence-associated genes were induced (Tosti et al. 2006). clear. According to Dong (2004) it could be obtained by The complex chain of events involved in O3 perception either activating the expression of genes coding for detoxi- and downstream signal transduction has been clearly des- fying enzymes such as GST induced by SA or by increasing cribed by Kangasjärvi et al. (2005). Ozone enters plant tis- the reducing power of the cell via the induction of the sues through the stomata and gains access to the apoplastic pentose phosphate pathway through the activation of the fluid surrounding the leaf cells where its degradation forms key enzyme glucose-6-phosphate 1-dehydrogenase induced ROS that are attacked by antioxidants such as ascorbate. by pathogen infection. Fobert and Després (2005) have also When the level of apoplastic ROS exceeds the detoxifying proposed that the redox changes of NPR1 and the TGA capacity of scavengers, two independent signal transduction transcription factors could be modulated by oxidoreductases chains are activated, one depending on heterotrimeric G- such as thioredoxins and glutaredoxins. proteins and the other on a MAPK cascade. The former in- Finally, ROS have been proposed to have a direct role duces ROS production in the chloroplast and subsequently in the establishment of SAR. Alvarez et al. (1998) have activates a plasma membrane respiratory burst NAPDH oxi- shown that the systemic signal for the induction of SAR in dase, enhancing the production of ROS in cells adjacent to tissues distant from the infection site could be the produc- the lesion. The MAPK cascade on the other hand could be tion of ROS. They have observed that in A. thaliana plants involved in the up-regulation of the biosynthesis of ET, inoculated with an avirulent strain of the bacterium P. syrin- which cooperates with SA in the perception and transduc- gae the production of ROS and H2O2 is not restricted to the tion of the ROS signal to nearby cells (Overmyer et al. inoculation site where the HR occurs, but can be detected 2003; Kangasjärvi et al. 2005). Lesion containment depends also at distant sites as systemic micro-oxidative bursts that on the synthesis of JA, based on lipid peroxidation products initiate SAR development. The systemic generation of ROS released as a consequence of cell death, which blocks ethy- by the micro-oxidative bursts could function in the estab- lene signaling most likely at the level of hormone percep- lishment of SAR activating the defense response throughout tion. A role in limiting lesion growth has been proposed also the plant, albeit at low levels, through the production of less for SA that inhibits the ET-biosynthetic enzyme ACC oxi- reactive second messengers, such as H2O2 and JA. However, dase. Both activation of MAPK and NADPH oxidase are the role of H2O2 as a potential second messenger has been dependent on intracellular calcium increase that takes place questioned by other studies (Dorey et al. 1998; Torres et al. in the early stages of signal transduction, probably via the 2006). oxidative activation of calcium channels and changes in the protein phosphorylation pattern with MAPK cascades again ROS and abiotic stress: the response to ozone playing a key role (Baier et al. 2005). Finally, abscisic acid (ABA) has a role in the regulation of the stomatal state and A detailed description of the role of ROS in the plant’s res- therefore of O3 influx to leaves (Torres and Dangl 2005; ponse to abiotic stress is beyond the scope of this review, as Kangasjärvi et al. 2005). The occurrence of stomatal clo- our main focus is the involvement of ROS in biotic stress. sure upon exposure to ozone is a well known phenomenon However we will take into account oxidative stress caused and the involvement of ABA in this process has been de- by ozone (O3) being particularly relevant per se as an exam- monstrated, probably with ROS functioning as interme- ple of abiotic stress and in the present context because the diates. acute, high-level exposure to this air pollutant induces a The common features of the response to ozone and pa- series of events that are typical of the pathogen defense res- thogens underscore a general motif in the stress response in ponse (Sandermann et al. 1998). Ozone is in fact considered that the signal transduction pathways for different stimuli a sort of abiotic “elicitor” of the defense response. often converge on common effectors and activate overlap- The effect of ozone on plants depends on a series of ping sets of genes (Wan et al. 2002 and references therein). variables i.e. the plant species or cultivar, the ozone concen- ROS, including the ROS-generating NADPH oxidase, tration and the duration of the exposure (Sharma and Davis MAPK cascades and lipid-derived signal compounds have 1994; Baier et al. 2005). Chronic exposure to low ozone been identified as major “points of convergence” for dif- levels reduces plant growth and crop yield without visible ferent plant responses (Mithöfer et al. 2004; Fujita et al. foliar damage, an effect generally attributed to enhanced 2006). Besides being involved in ROS production in hyper- respiration, reduced transpiration and photosynthesis, and sensitively reacting cells, the Arabidopsis AthbohD and premature senescence, while high levels of ozone lead to AthbohF genes are also expressed in stomatal guard cells 2+ the appearance of necrotic lesions and/or chlorotic symp- where H2O2 induces an ABA-mediated increase of Ca in toms on leaves. A biphasic oxidative burst involving a the cytoplasm leading to stomatal closure (Torres and Dangl NADPH-dependent oxidase (Rao and Davis 1999) and re- 2005). As a further confirmation of the interplay between sulting in the generation and accumulation of H2O2 and these two pathways, fungal elicitors have been shown to in- superoxide anion has been proposed to be the main mecha- duce both ROS and stomatal closure by the same mechanism for ozone-induced cell death (Langebartels et al. nism (Klusener et al. 2002). These same genes seem also to 2002). A clear spatial and quantitative correlation between be responsible for the generation of ROS signals in the res- the accumulation of ROS and ozone damage has in fact ponse to ozone stress (Joo et al. 2005). Finally, signal trans- been found in several plant species, including Arabidopsis, duction systems based on PLD and PA take part as well in tobacco, tomato and birch (Wohlgemuth et al. 2002; Over- the response to several abiotic stress conditions such as myer et al. 2003). The study of ozone-induced gene expres- metal excess, drought, cold or wounding (Navari-Izzo et al. sion has revealed that the ozone-dependent gene induction 2006 and references therein). overlaps to a certain extent with that observed during the

35 ROS in the plant stress response. Bettini et al.

CONCLUSIONS an aspect even more pronounced in plant-pathogen interact- tions where a continuous process of coevolution takes place. Reactive Oxygen Species are clearly emerging as a leitmotif If the widespread adoption of a few model species has in plant life, being involved in most physiological responses allowed to obtain an increasingly clear picture of the com- to stress as well as in developmental processes such as sto- mon features underlying these responses, only the study of matal closure, root elongation and cell expansion (Lamb diverse plant systems will help in understanding the mul- and Dixon 1997; Grant and Loake 2000; Overmyer et al. tiple facets of a fascinating phenomenon. 2003; Apel and Hirt 2004; Laloi et al. 2004). It is therefore not surprising that at least 152 genes are comprised in the ACKNOWLEDGEMENTS so-called “reactive oxygen gene network” in the Arabidop- sis genome, involving multiple enzymes for ROS produc- Authors are grateful to Prof. A. Scala (Department of Agricultural tion and scavenging (Mittler et al. 2004). Biotechnology, University of Florence, Italy) for helpful discus- One of the main points is that the generation of active sion on the manuscript. oxygen species is a key process shared between the response to both biotic and abiotic stress where ROS can either REFERENCES seriously damage cells or be interpreted as a signal for the Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen species and activation of downstream responses. The outcome of the antioxidants: relationships in green cells. Physiologia Plantarum 100, 224- oxidative stress depends probably on the existence of a 233 threshold in the level of ROS accumulated as a result of Alvarez ME, Pennell RI, Meijer P-J, Ishikawa A, Dixon RA, Lamb C stress conditions. 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