Hydrogen peroxide—a central hub for information ﬂow in ...

Open access – Invited reviewTHIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED‘MOLECULAR BASIS OF PLANT STRESS’

Hydrogen peroxide—a central hub for information flowin plant cellsVeselin Dimitrov Petrov1,2,3 and Frank Van Breusegem2,3*1 Department of Plant Physiology and Plant Molecular Biology, University of Plovdiv, 24 Tsar Assen str., Plovdiv 4000, Bulgaria2 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium3 Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium

Received: 26 January 2012; Returned for revision: 4 March 2012; Accepted: 14 April 2012; Published: 18 April 2012

Citation details: Petrov VD, Van Breusegem F. 2012. Hydrogen peroxide—a central hub for information flowin plant cells. AoB PLANTS 2012: pls014; doi:10.1093/aobpla/pls014

Abstract

Background Hydrogen peroxide (H2O2) was initially recognized as a toxic reactive oxygen species, able tocause damage to a variety of cellular structures. However, it became clear in the last decadethat H2O2 can also act as a potent signalling molecule, involved in a plethora of physiologicalfunctions.

Scope In the present review, we offer a brief summary of H2O2 signalling events and focus on themechanisms of its perception and signal transduction, the factors that act downstream, aswell as H2O2 interference with other information transfer mechanisms.

Conclusion The significant scientific effort in the last 10 years to determine the position of H2O2 in signaltransduction networks in plants demonstrated that it is essential for both the communicationwith external biotic and abiotic stimuli and the control of developmentally regulated pro-cesses. In addition, H2O2 complements, synergizes or antagonizes many cellular regulatorycircuits by active interaction with other signals and plant hormones during growth, develop-ment and stress responses. Therefore, further understanding of H2O2 signal transduction isnot only of fundamental, but also of practical importance, since this knowledge may contrib-ute to improve agricultural practices and reduce stress-induced damage to crops.

IntroductionReactive oxygen species (ROS) are produced either afterincomplete reduction of oxygen (hydrogen peroxide—H2O2; superoxide radical—O2

2; hydroxyl radical—HO·)(Gechev et al. 2006) or energy transfer to its chemicallyinert triplet ground state (singlet oxygen—1O2) (Kim

et al. 2008). These oxygen derivatives possess a strongoxidizing potential that leads to damage to a variety ofbiological molecules and are therefore unwelcomebyproducts of normal metabolic processes in allaerobic organisms (Halliwell 2006). During periods ofbiotic or abiotic stress, ROS levels can rise excessively,leading to an oxidative stress state (Apel and Hirt

* Corresponding author’s e-mail address: [email protected]

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2004). Since plants are sessile organisms and cannotsimply escape from adverse environmental conditions,they have developed an elaborate system to control cel-lular ROS concentrations (Mittler et al. 2011). In addition,plants have evolved a way to utilize lower concentrationsof ROS as signalling molecules for a number of regulatedprocesses during plant growth and development, like cellelongation (Foreman et al. 2003) and differentiation(Tsukagoshi et al. 2010), as well as in responses to avariety of environmental stimuli (Dat et al. 2000;Gapper and Dolan 2006).

Among the ROS compounds, H2O2 is the one thatreceived most of the attention of the scientific commu-nity in the last decade. Hydrogen peroxide is the resultof a two-step reduction of molecular oxygen (the firststep leading to superoxide radical) and has a relativelylong lifespan in comparison to other ROS. The long half-life (1 ms) of H2O2 and its small size allow it to traversecellular membranes and migrate in different compart-ments, which facilitates its signalling functions (Bienertet al. 2006). As a result, it is now well proved that H2O2

is a regulator of a multitude of physiological processeslike acquiring resistance, cell wall strengthening, senes-cence, phytoalexin production, photosynthesis, stomatalopening and the cell cycle. The multi-functionality on theone hand, and the danger it presents in elevated con-centrations on the other hand, require the very strictcontrol of H2O2 concentration in plant cells.

Active production H2O2 occurs mostly at the apoplasticspace and is required for triggering the ‘oxidative burst’that is a part of the hypersensitive response to patho-gens, but is also a prerequisite for normal growth, devel-opment and cell death (Miller et al. 2010). The mainsource of this H2O2 is a class of cell membrane NADPH-dependent oxidases like respiratory burst oxidase homo-logues (Rboh), which are regulated by a unique class ofRho-like proteins called ROPs (Rho-related GTPasesfrom plants) (Agrawal et al. 2003), as well as cellwall-associated peroxidases (Bolwell et al. 2002). Ofcourse, multiple other sources of H2O2 exist in differentplant cell compartments, but these are the result ofincreased metabolism (like photorespiration and fattyacid oxidation in peroxisomes and glyoxisomes, as wellas overenergization of the electron transport chains inchloroplasts and mitochondria, etc.) (Fig. 1). In mostcases, H2O2 is formed after reduction of superoxide radi-cals catalysed by superoxide dismutase (SOD). Simultan-eously, a vast network of antioxidants is constantly onthe alert for rising H2O2 concentrations and provideseffective scavenging for it (Apel and Hirt 2004; Gechevet al. 2006; Miller et al. 2010). This antioxidant systemconsists of several enzymes, such as catalase (CAT), as-corbate (APX) and secretory peroxidases (POX),

glutathione reductases (GR) and peroxiredoxines (Prx),and non-enzymatic compounds like tocopherols, ascor-bic acid and flavonoids (Willekens 1995; Noctor andFoyer 1998; Asada 1999; Miller et al. 2010).

The biological effect of H2O2 is mostly dependent onits concentration, but also on the site of production,the developmental stage of the plant and previous expo-sures to different kinds of stress. Generally, at low con-centrations it acts as a signalling molecule, while athigher concentrations it provokes the onset of celldeath (Gechev and Hille 2005). Hydrogen peroxide-induced cell death is essential for some developmentalprocesses and environmental responses, including aleur-one cell death, leaf senescence, hypersensitive responseto pathogens, allelopathic plant–plant interactions,etc. (Bethke and Jones 2001; Bais et al. 2003; Gechevet al. 2005).

It is obvious that H2O2 is a molecule with enormousimpact on normal plant cell functioning. Althoughmuch progress has been achieved in revealing its rolein plants, there is still a lot to be unveiled. Yet, in thisreview we focus on the present state of the art in knowl-edge concerning H2O2 and its signalling network.

H2O2 signalling network

Perception of H2O2 signals

A wide range of environmental stimuli lead to a transientrise in cellular H2O2 levels. In these cases, H2O2 can beviewed as a signal that relays the initial stimuli to down-stream effectors. The existence of H2O2 sensing proteinsstill remains enigmatic. However, it is presumed thatincreased H2O2 levels could be perceived directly byredox-sensitive transcription factors (TFs) that orches-trate downstream cascades (Miller et al. 2008). Goodcandidates for such TFs are class A heat shock factors(Hsfs), which are shown to be responsive to oxidativestress both in animals and plants (Miller and Mittler2006; Kotak et al. 2007). Moreover, it was demonstratedthat Hsfs of both Drosophila and mammalian origin areable to interact directly with H2O2, and as a result formDNA-binding-competent homotrimers in a reversiblemanner (Zhong et al. 1998; Ahn and Thiele 2003).Plants may utilize a similar mechanism to detect H2O2

signals.In addition, a recent study suggested another intri-

guing alternative for H2O2 perception in planta: the oxi-dation of methionine (Met) to methionine sulphoxide(MetSO) may couple oxidative signals to changes in theprotein phosphorylation state (Hardin et al. 2009). Theauthors provide in vitro and in vivo evidence that Met oxi-dation by H2O2 may compromise peptide phosphoryl-ation when these residues are situated within

2 AoB PLANTS 2012: pls014; doi:10.1093/aobpla/pls014, available online at www.aobplants.oxfordjournals.org & The Authors 2012

Petrov and Van Breusegem — Hydrogen peroxide is a hub for information flow in plants

phosphorylation motifs. As an example, the in vitro oxi-dation of Met538 in leaf nitrate reductase (NR) stronglyinhibits the phosphorylation of the adjacent Ser534.Moreover, exogenously applied H2O2 causes a reductionof Ser534 phosphorylation in vivo as well, while the over-expression of a cytosolic MetSO repair enzyme has the

opposite effect and leads to an increase in phosphoryl-ation rates. Such a redox methionine-dependent switchwould be a fast and efficient way to relay the oxidativesignal further downstream. Future research on thetopic should identify and catalogue proteins with suit-able target Met residues in order to unravel how

Fig. 1 The main mechanisms of H2O2 synthesis and catabolism in different cellular structures. The apoplast is a space of active ROSproduction, due to the activity of NADPH-dependent oxidases (Rboh), cell wall peroxidases, amine oxidases and oxalate oxidases (thelatter two are not included in the figure). Superoxide anions which result from the action of these enzymes are readily converted toH2O2. Apoplastic H2O2 serves not only to induce oxidative burst during pathogen attack, but also to regulate cell wall rigidity. Insidethe cell, the compartments that produce the highest amounts of ROS are the chloroplasts. Hydrogen peroxide is an undesirable by-product of photosynthesis and a number of scavengers can be found in the chloroplasts that are able to disarm it by turning it into harm-less water molecules. These include members of the glutathione-ascorbate cycle and peroxiredoxins/thioredoxins. Another site of activeROS synthesis is the mitochondria. In plants, excessive ROS accumulation in mitochondria can be alleviated by an alternative mechanismfor electron transport which is carried out by alternative oxidase (AOX). Although processes like photorespiration and fatty-acid oxidationgenerate H2O2 in peroxisomes and glyoxisomes, dangerous raising of H2O2 is normally prevented by the presence of the enzyme cata-lase. Finally, H2O2 and other ROS that leak out in the cytoplasm are easily handled by the activity of ROS-scavenging systems similar tothose in the chloroplast.



widespread this mechanism could be. A solution toachieve this is offered by a recently developed proteomictechnology called COFRADIC (combined fractional diag-onal chromatography) which, among its other usefulapplications, is able to identify oxidized methionine resi-dues and quantify their degree of oxidation. In the firststudy using this new method, COFRADIC analyses onhuman Jurkat cells revealed that H2O2-sensitive methio-nines are preferentially located in zones exposed to theprotein surface, enriched for charged and poor in hydro-phobic residues (Ghesquiere et al. 2011).

H2O2 transport

One important feature that facilitates H2O2 in its inter-mediary functions is its ability to diffuse across mem-branes. It was shown, for example, that H2O2 producedby the chloroplast electron transport chain can leakout of chloroplasts in a light-intensity-dependentmanner (Mubarakshina et al. 2010). However, H2O2 is arelatively neutral solute and native membranes presenta significant barrier to its free diffusion (Bienert et al.2006). Hydrogen peroxide can be transported throughspecific membrane aquaporin homologues of the TIP(tonoplast intrinsic protein) and PIP (plasma membraneintrinsic protein) families. For instance, it was shown thatexpression of Arabidopsis thaliana AtTIP1;1 and AtTIP1;2genes in yeast cells decreased their survival rate in thepresence of H2O2, while blocking this aquaporin-mediated diffusion alleviated the effect of H2O2

(Bienert et al. 2007). Moreover, a more recent analysissuggests that the Ar/R (aromatic/arginine) regions inPIP2 proteins are critical for their selectivity towardsH2O2 (Dynowski et al. 2008) and as all eight PIP2 proteinsin Arabidopsis are conserved in these positions, presum-ably all of them are involved in the specific transportof H2O2.

In turn, H2O2 is able to influence the transport ofwater and other solutes between cells. In Phaseolus vul-garis it was found that H2O2 exerts concentration-dependent effects on root hydraulic conductivity, whichis one of the determinants of root water uptake. Levelsof H2O2 ,1 mM increase conductivity, while those.1 mM decrease it (Benabdellah et al. 2009). In Arabi-dopsis root meristems, H2O2 treatments induce modifi-cations of plasmodesmal flux based on H2O2 amountsin a similar manner—low concentrations boost plasmo-desmal permeability while high concentrations inhibit it(Rutschow et al. 2011). An attractive, but still speculativeway to explain these phenomena is that low levels ofH2O2 create a signal for mild stress, which can be alle-viated by increased flow of solutes and nutrients. Con-versely, accumulation of higher amounts of H2O2

produces a message for severe stress, like during

pathogen invasion, which requires that the cell is iso-lated (Rutschow et al. 2011).

H2O2 as a second messenger

Hydrogen peroxide possesses some features typical forsecond messenger molecules. For example, its produc-tion is easily upregulated by many stimuli, mainlythrough NADPH-oxidases and peroxidases, as alreadydiscussed. In addition, H2O2 is a small and relativelymobile molecule that has the potential to carry informa-tion between different cellular compartments. Moreover,H2O2 is able to modulate the activities of many other sig-nalling components (thus relay signals) and intercalatein a number of signalling cascades with different bio-logical outcomes, including the one that leads to itsown synthesis (Fig. 2). In the latter case, either a positiveor a negative feedback is provided by inducing or inhibit-ing H2O2 modulating systems (Mittler 2002; de Pintoet al. 2006; Van Breusegem and Dat 2006). This ismainly dependent on the H2O2 concentration and thetiming of its synthesis. The possibility of a positive feed-back provides a way to amplify the initial signal (anotherimportant characteristic of second messengers), whilethe negative feedback option ensures that the systemcan be effectively switched off in order to prevent exces-sive damage.

The most typical targets of H2O2 include effectors ofcalcium homeostasis, ion channels, protein kinases orphosphatases and TFs.

Mitogen-activated protein kinases and H2O2

Mitogen-activated protein kinases (MAPKs) in plantsform a large network implicated in a vast array of func-tions, including the relay of H2O2 signals (Zhang et al.2006; Xing et al. 2008). Mitogen-activated proteinkinases can either be activated by H2O2 accumulation,or trigger an H2O2-induced oxidative burst themselves(Nakagami et al. 2005; Pitzschke et al. 2009). Thus,while H2O2 activates the ANP1 (an MAPK kinase kinase)protein, which in turn initiates a phosphorylationcascade with AtMPK3 and AtMPK6 (Kovtun et al. 2000),the MEK2 pathway is part of an amplification mechan-ism upstream of Rboh genes, which are known toproduce H2O2 (Yoshioka et al. 2003). This positioning ofMAPK cascades up- or downstream of H2O2 indicatesthe complexity and multidirectionality of stress signal-ling networks.

A prominent place among the module effectorsmodulated by ROS metabolism in Arabidopsis holdsthe MEKK1–MKK1/2–MPK4 pathway. The MAPK kinasekinase MEKK1 is regulated by different stresses andH2O2 in a proteasome-dependent manner (Pitzschkeet al. 2009). MEKK1 can activate the downstream



kinases MPK3, MPK4 and MPK6. However, only mpk4mutants possess a similar phenotype to mekk1 andhave similar changes in gene expression. Therefore, itis MPK4 that is usually considered as an H2O2-inducedMEKK1 target. The signal between MEKK1 and MPK4 istransmitted by the MAPK kinases MKK1 and MKK2 (Qiuet al. 2008). These are in part functionally redundant,as single mkk1 or mkk2 mutants have normal activitiesof MPK4, unlike the double mkk1/mkk2 mutant (Gaoet al. 2008). Analyses of gene expression reveal thatof 32 TFs that are highly responsive to multiple ROS-generating conditions, 20 are regulated via theMEKK1–MKK1/2–MPK4 pathway (Pitzschke et al.

2009). This confirms the central role of these factorsin oxidative stress signalling. Surprisingly, a uniqueshortcut in MAPK pathways has been found forMEKK1—it can interact directly with the TF WRKY53and phosphorylate it (Miao et al. 2007). This modifica-tion increases WRKY53 DNA-binding activity and allowsthe immediate induction of stress and defence-relatedtarget genes by bypassing of downstream kinases. Inaddition, MEKK1 was demonstrated to possess theability to bind WRKY53 promoter in a region whichparticipates in the switch from a leaf-age-dependentto a systemic plant-age-dependent WRKY53 expression(Miao et al. 2007).

Fig. 2 A simplified schematic representation of the major signalling components in the H2O2-transduction network, their interactionsand different outcomes in the plant cell. Stress conditions and a variety of environmental cues lead to accumulation of H2O2, whichserves as a second messenger and relays the signal to downstream effectors, including TFs, MAP-kinases, miRNAs, etc. Some ofthese effectors can in turn interact with each other. The information flow in this network is rarely straightforward, as it diverges insome points and converges in others. The final output of the cascade depends on the nature of the signal, H2O2 concentration, locusof H2O2 synthesis, interaction with other active signalling pathways, previous exposure to stress, etc.



In plants, transmission of pathogen and diseasesignals is also governed by MAPKs in a H2O2-dependentmanner. This function is exerted in tobacco by SIPKand WIPK kinases (Asai and Yoshioka 2008), whereasin Arabidopsis it is exerted by their homologues MPK3and MPK6 (Nakagami et al. 2005). MPK3/6 are not onlyactivated by ANP1, as mentioned above, but also bythe serine–threonine kinase OXI1, which similarly canbe induced directly by H2O2 (Rentel et al. 2004). Hydro-gen peroxide may also be involved in MPK3/6 responsesto heavy metals, since these two MAPKs are activatedafter cadmium (Cd) treatment by a mechanism that pre-sumably requires accumulation of H2O2 (Liu et al. 2010).Recently, it has been proved that the MAPK phosphataseMPK2 can functionally interact with MPK3 and MPK6(Lumbreras et al. 2010). By dephosphorylating regulatoryresidues in these targets, MPK2 provides a negativesignal with profound effects on pathogen reactions, asshown by mpk2 mutant phenotypes. Moreover, MPK2seems to have differential functions in biotrophicversus necrotrophic responses, as mpk2 plants exhibitopposite symptoms when infected with the biotrophicRalstonia solanacearum or the necrotrophic Botrytiscinerea. mpk2 mutants are more resistant toR. solanacearum and more susceptible to B. cinerea, sug-gesting that MPK2 is a negative regulator of theHR-induced (hypersensitive response) cell death whichprotects against biotrophic pathogens but facilitatesnecrotrophic infections.

Two recent studies show that brassinosteroids (BRs)are able to provoke apoplastic H2O2 accumulation,which further switches on MPKs to increase stress toler-ance in different species. In cucumber, Rboh, MPK1 andMAPK3 are among the target genes that are upregulatedin this BR-induced process (Xia et al. 2009); in maize,BR-stimulated ZmMPK5 induction drives an NADPHoxidase-mediated amplification loop required for the en-hancement of the activities of antioxidant enzymes(Zhang et al. 2010).

Transcription factors acting downstream of H2O2

The last link in the chain of H2O2 signal mediators is avariety of TFs. They induce a massive reprogrammingof the transcriptome and lead to a second wave of effec-tors, which in turn switch on various responses likedefensive mechanisms and the assembly of the celldeath machinery. The TFs that are associated withH2O2 signalling are members of different families, in-cluding NAC, ZAT, WRKY, DREB, bZIP and MYB.

In Arabidopsis, many NAC TFs control leaf senescence.Notably, 15 senescence-associated NAC TFs are upregu-lated by H2O2 (Balazadeh et al. 2010a), of which

ANAC032 and ANAC042 are the most significantlyinduced (around 25 times). ANAC042 has already beenreported previously as a possible target of H2O2 path-ways (Gechev et al. 2005) and it was proposed that itsactivation occurs through the OXI1/MPK3/6 cascade.Another key regulator of age- and salt-promoted senes-cence is ANAC092. As 40 % of its salt-dependent senes-cence regulon is actually affected by H2O2, an oxidativeburst obviously plays a role in ANAC092-mediatedresponses (Balazadeh et al. 2010a, b). More recently, asimilar regulatory network, which includes cross-talkbetween salt and H2O2, was also ascribed to ORS1, theparalogue of ANAC092 (Balazadeh et al. 2011). Moreover,the authors even localized a H2O2-responsive regulatoryelement in the proximal 230 bp of the ORS1 promoterregion (Balazadeh et al. 2011).

The zinc-finger TFs from the Zat family are also oftenrelated to oxidative stress. For example, Zat11 andZat12 are highly induced by ROS (Gadjev et al. 2006).In addition, overexpression of Zat12 leads to increasedlevels of oxidative stress-responsive transcripts, whileits inhibition results in higher sensitivity to H2O2

(Rizhsky et al. 2003; Davletova et al. 2005). In experi-ments with apx1 mutants, Miller et al. (2008) nominatedthree Zat TFs as being involved in ROS signalling andabiotic stress–Zat7, Zat10 and the already mentionedZat12. Remarkably, the induction of Zat7 by H2O2 isimpaired in knockout Zat12 plants, which suggests adirect dependence of Zat7 expression on Zat12.However, this relationship is probably more complex, asZat12-overexpressing plants fail to modulate Zat7expression.

WRKY TFs are often described as key players in plantresponses to biotic, abiotic and oxidative stresses(Eulgem and Somssich 2007). Of these TFs, WRKY52 andWRKY70 are the most widely discussed H2O2-induciblerepresentatives of the family. WRKY52 is a senescence-related factor which can affect the expression of avariety of stress, defence and senescence-associatedgenes. Its overexpression leads to accelerated senes-cence, while its knockout provokes a delay in thisprocess (Miao et al. 2004). On the other hand, WRKY70 isspecific for salt stress and shows a similar expressionpattern to Zat7 in apx1 mutant plants (Miller et al. 2010).It was previously demonstrated that WRKY70, togetherwith the HASTY TF, and Zat7 actually interact in a yeasttwo-hybrid assay through the EAR-domain of Zat7(Ciftci-Yilmaz et al. 2007).

Other H2O2-induced TFs from different families includeDREB2A that are upregulated not only by oxidativestress, but also by drought, cold and heat stress(Rizhsky et al. 2003), many bZIP, ERF and MYB factors



that configure the response of japonica rice to chillingstress (Yun et al. 2010), and the redox-sensitive Hsfswhich were described above as possible direct sensorsof H2O2.

miRNAs and H2O2

Plant miRNAs are known to be ubiquitous regulators ofgene expression at the post-transcriptional level andmodulate a great diversity of biological responses bydirecting mRNA targets for cleavage or inducing transla-tional silencing. Recently, in the first genome-wide studyof H2O2-regulated miRNAs in plants, seven miRNA fam-ilies were found to be differentially expressed afterH2O2 treatment in rice seedlings (Li et al. 2011). Theseinclude miR169, miR397, miR528, miR827, miR1425,miR319a.2 and miR408-5p. Five of these miRNA familiesare upregulated by H2O2 (miR169, miR397, miR827,miR1425 and miR408-5p) and two are downregulated(miR528 and miR319a.2). Among the direct targets ofthese miRNAs fall: a HAP2-like TF for miR169; laccasesfor miR397 (presumably involved in lignin biosynthesis);pentatricopeptide repeat proteins for miR1425 (import-ant for organellar biogenesis); major facilitator super-family proteins for miR408-5p and miR827 (a groupof secondary transporters); putative IAR1 proteins(IAA-alanine resistance protein 1), known to be import-ant for auxin homeostasis, for miR528; and a putativemetacaspase for miR319a.2.

Other H2O2-induced factors

The oxidative stress resulting from different environmen-tal cues also leads to the modulation of many effectorsthat help to carry out adaptive changes of cellular physi-ology. A convenient system to study the effects of en-dogenous peroxisome-produced H2O2 is presented bycat2 knockout plants (Queval et al. 2007). This modelwas used to identify defence-related components thatare controlled by H2O2. One such modulator in Arabidop-sis is the early H2O2-responsive UDP-glucosyltransferaseUGT74E2, which influences the water stress responseand plant architecture by its preference for auxinindole-3-butyric acid (IBA) as a substrate for glycosyla-tion (Tognetti et al. 2010). UGT74E2 is a gene thatresponds with a drastic boost of expression(.100-fold) in cat2 mutants subjected to high-lighttreatment. Genetic analyses with plants ectopicallyexpressing UGT74E2 indicated that IBA and indole-3-acetic acid (IAA) homeostasis were altered, whichresulted in structural changes like increased shoot branch-ing and compressed rosette structure, as well as in signifi-cantly improved survival during drought and salt stress.Hence, the action of this UDP-glucosyltransferase pre-sents a link in the interplay between H2O2 and auxin

pathways. In another recent study with cat2-deficientplants, it was demonstrated that myo-inositol (MI) levelsare seriously decreased, independently of salicylic acid(SA) accumulation. These data indicate a role of MI incell death triggered by peroxisomal H2O2 and suggestthat its cellular concentration is a major factor that deter-mines the outcome of defence responses to oxidativestress (Chaouch and Noctor 2010).

The Arabidopsis peptide kiss of death (KOD) is anotherH2O2-inducible regulator of programmed cell death(PCD) (Blanvillain et al. 2011). It is induced nine-foldafter application of 30 mM H2O2 to leaf discs and its ex-pression is linked to caspase3 activity, a strong hallmarkof PCD. KOD is also involved in embryo development asits mutation reduces PCD of the suspensor cells (Blanvil-lain et al. 2011). Although the mode of action of KOD isstill unclear, it is the first short open reading framepeptide reported to be involved in PCD regulation.

Earlier this year, a group in Tsinghua University per-formed the first apoplast proteome investigation ofplant seedlings in response to H2O2 (Zhou et al. 2011).The authors propose a protein network that providesclues for oxidative stress responses in rice root apoplasts.Basically, the 54 differentially regulated H2O2-responsiveproteins described in this study could be divided into twocategories: related to redox regulation and related tocellular metabolism. The first group includes 24 proteinswhich sense stress signals and modulate redox state:many class III peroxidases, malate dehydrogenase,putative receptor-like proteins (RLPs), thiol redox-associated proteins (FMO and PDI), a chitinase and apathogenesis-related protein (PR-1a). The second groupis needed to adjust the metabolic flows in order to alle-viate the deleterious effects of stress. Remarkably, 26 ofall the reported proteins are involved in carbohydratemetabolism and 17 of these are glycosylhydrolases(GHs). Most of the latter are downregulated after H2O2

treatment, which may lead to rearrangements of cellwall constituents. On the other hand, enzymes likeUDP-glucose pyrophosphorylase, pectinesterase anda-arabinofuranosidase are upregulated, and maycontribute to strengthening the cell wall by influencingpolysaccharide turnover and increasing pectindemethylesterification.

H2O2 interference with signallingpathwaysHydrogen peroxide communicates with a variety of othersignalling molecules and plant hormones. Together theyform an intricate network that is still not completelycharacterized. Here we present some details on theinteraction of H2O2 with Ca2+, SA and nitric oxide (NO).



H2O2 and Ca21

Like H2O2, Ca2+ is also a ubiquitous second messengerable to orchestrate different physiological reactions.There are numerous data in the literature indicatingthat H2O2 and Ca2+ homeostasis are interdependent,with some cases in which H2O2 regulates Ca2+ ionfluxes, while in others Ca2+ affects H2O2 metabolism. Aclassic example of H2O2-mediated Ca2+ signalling isthe cascade leading to stomatal closure in response todrought. In this chain of reactions, the plant hormoneabscisic acid (ABA) induces the production of H2O2 inguard cells, which results in the opening of Ca2+-permeable channels and an increase in the cytoplasmicCa2+ level ([Ca2+]cyt) in intact guard cells (Pei et al. 2000).Moreover, if H2O2 production is blocked, ABA-inducedclosure of stomata is inhibited. In Arabidopsis seedlings,the application of H2O2 triggers a biphasic Ca2+ eleva-tion, with the first peak located in cotyledons and thesecond in the root (Rentel and Knight 2004). Important-ly, the short time needed before the first Ca2+ rise sug-gests that Ca2+ influx may be among the earliestresponses to H2O2. The delay in the second calciumpeak indicates that H2O2 may catalyse a [Ca2+]cyt riseby specific mechanisms in different tissues. More recent-ly, it was shown that spermidine oxidase-derived H2O2

regulates pollen plasma membrane hyperpolarization-activated Ca2+ channels in order to induce pollen tubegrowth (Wu et al. 2010). Pollen from polyamine oxidase(PAO) mutants was unable to cause the opening of Ca2+-permeable channels in the presence of spermidine,which resulted in reduced pollen tube growth and seednumber.

Cytosolic Ca2+ is in turn able to trigger changes in H2O2

concentration. Hydrogen peroxide synthesis requires acontinuous Ca2+ influx which activates NADPH oxidaseslocated at the plasma membrane (Lamb and Dixon1997). Experiments with Arabidopsis challenged withPseudomonas syringae demonstrated that the NADPHoxidase inhibitor diphenylene iodonium does notchange the Ca2+ balance, but the Ca2+ channel blockerLaCl3 suppresses H2O2 accumulation and the hypersensi-tive response (Grant et al. 2000). Thus, in this case it isagain Ca2+ that acts upstream of H2O2. Similarly, itappears that in tobacco cells, the free sphingoid LongChain Base (LCB) sphinganine induces transient increasesin the nuclear and cytosolic Ca2+ concentration and thedownstream production of H2O2 (Lachaud et al. 2011).Interestingly, according to the model of sphinganine sig-nalling proposed by the authors, only the nuclear Ca2+

flow is implicated in subsequent H2O2-independent celldeath mechanisms, while the [Ca2+]cyt current is neces-sary for H2O2-induced basal cell defence responses.

Therefore, it seems that in this model system, cell deathis uncoupled from H2O2 pathways.

The antioxidant system may also be a target of Ca2+

influence. For example, the efficiency of H2O2 scavengingin Arabidopsis plants depends on the peroxisomal Ca2+

concentration (Costa et al. 2010). It appears that theintracellular Ca2+ rise significantly accelerates H2O2 de-toxification and this is achieved at least in part withthe help of the Ca2+-sensitive catalase CAT3. Calmodulin(CaM) is a ubiquitous Ca2+-binding protein, which canregulate a number of different protein targets, therebyaffecting many different cellular functions (Chin andMeans 2000). One of its targets is the MAP kinase 8(MPK8) which is mainly operative after mechanicalwounding. Takahashi et al. (2011) recently proved thatthe MPK8 pathway in turn negatively regulates H2O2 syn-thesis by controlling the expression of RbohD.

Calcium-dependent protein kinases (CDPKs) sense theCa2+ concentration changes in plant cells and play im-portant roles in signalling pathways for disease resist-ance and a number of stress responses. In some cases,biotic and abiotic stresses are mediated by CDPKs,which are upregulated after an H2O2 burst. In tomato,LeCDPK1 expression is rapidly and transiently enhancedin leaves treated with H2O2 (Chico et al. 2002). Similarly,in wheat, eight out of 20 studied CDPKs respond to H2O2

treatment (Li et al. 2008).

H2O2 and SA

Salicylic acid is a key signalling molecule in plantsexposed to biotrophic pathogens (Pieterse et al. 2009),required for the establishment of the systemic acquiredresistance (SAR) response (Dempsey and Klessig 1994).Hydrogen peroxide can be linked directly to SA, sincebenzoic acid (the immediate precursor of SA) is con-verted into SA by the H2O2-mediated activation ofbenzoic acid 2-hydroxylase (Leon et al. 1995). At thesame time, SA also has the capacity to enhance the en-dogenous level of H2O2, mainly by inducing the activa-tion of SOD (Rao et al. 1997). Thus, according to thisscenario H2O2 and SA can work together as a self-amplifying system.

In cat2 plants, which were mentioned earlier, theeffect of oxidative stress also relies on isochorismatesynthase 1 (ICS1). This conclusion was drawn after thecat2 line was crossed with sid2, which is defective inICS1, and it was observed that lesion formation is com-pletely absent in the cat2 sid2 double mutants (Chaouchet al. 2010). This indicates that the isochorismatepathway of SA synthesis couples intracellular oxidativestress to cell death and disease resistance responses.The cat2 mutants were also used in a study that



addresses the role of glutathione reductase 1 (GR1) inH2O2 responses (Mhamdi et al. 2010). The authorscarried out analyses in cat2 gr1 double mutants andestablished that the GR1-dependent glutathione statusis decisive for multiple responses to an increased H2O2

concentration, including the accumulation of SA andthe subsequent induction of pathogenesis-relatedgenes. In turn, sid2 plants were utilized to investigateseed germination processes under high salinity (Leeet al. 2010). While the inhibitory effect of salt stress onsid2 germination is strengthened in the presence ofhigher SA concentrations, treatment with ,50 mM SAsignificantly alleviates it. This can be partially explainedby the fact that the elevation of endogenous H2O2

levels is hindered after treatment with moderate dosesof SA. The germination of sid2 seeds under such condi-tions of high salinity is hypersensitive to H2O2, but thephysiological concentrations of SA modulate antioxidantactivity to prevent oxidative damage (Lee et al. 2010).

Similar results were obtained in tomato, where theeffect of SA on root viability during NaCl treatments isalso concentration dependent (Gemes et al. 2011).Again, the lower quantities of SA reduce the accumula-tion of ROS and NO, and help the plant withstand thesalt stress, while the higher ones provoke an oxidativeburst and disorganization of the root meristem. Salicylicacid provokes ameliorative effects during heavy metalstress as well. In a similar manner, the exogenous appli-cation of SA relieves Cd toxicity by reducing H2O2 accu-mulation in root apoplasts of the legumes Phaseolusaureus and Vicia sativa (Zhang et al. 2011). Anexample of H2O2 antagonizing SA signalling is thenuclear translocation of the SAR-associated transcriptionactivator NPR1. In this case, cytoplasmic H2O2 wasshown to inhibit the SA-induced transport of NPR1to the nucleus and thus block the activation ofpathogenesis-related (PR) genes (Peleg-Grossman et al.2010).

These results demonstrate that the interactionsbetween H2O2 and SA are multi-faceted, and can varyfrom cooperation to mutual inhibition in different con-texts, concentrations and conditions.

H2O2 and NO

Nitric oxide is a second messenger in plants which dis-plays both pro-oxidant and antioxidant properties. Onthe one hand, NO is able to induce scavenging ofexcess H2O2 and thus inhibit peroxide signalling path-ways; on the other hand, NO may also collaborate withH2O2 to switch on SAR, or stress tolerance (Małolepszaand Rozlska 2005). For example, NO and H2O2 have over-lapping roles in the acclimation to salinity. Proteomicanalysis revealed that 85 proteins vary quantitatively in

citrus plants exposed to salt stress. However, pre-treatment with either H2O2 or sodium nitroprusside (aNO-releasing chemical) prevented changes in the accu-mulation levels of 45 of them and alleviated the detri-mental effects of high salinity (Tanou et al. 2009).Another example of H2O2 and NO exerting similar func-tions is their influence on the stimulation or inhibitionof root hair growth induced by different concentrationsof extracellular nucleotides in Arabidopsis (Clark et al.2010). In contrast, NO mediates tolerance to copper tox-icity in tomatoes, partly by antagonizing the H2O2 effect,as it efficiently leads to a boost of the antioxidantsystem activity (Wang et al. 2010a). Similarly, in maizeleaves ABA-induced H2O2 production leads in turn toNO generation, which activates MAPK pathways, tofinally result in the upregulation of the expression andactivities of antioxidant enzymes (Zhang et al. 2007).

It was demonstrated that synergistic interactionsbetween NO and H2O2 can be necessary for inductionof PCD. For example, in tobacco Bright Yellow-2 (TBY-2)cells, both molecules are required for the activation ofPCD and the addition of an NO scavenger restores thecell viability after treatment with H2O2 (de Pinto et al.2006). Interestingly, the NO synthesis is stimulatedonly after direct application of H2O2, but not after pro-longed exposure to H2O2, supporting the view that dis-tinct signalling mechanisms are activated underdifferent combinations of conditions. Similarly, insoybean cells, PCD can be triggered by high levels ofNO, but only when supplemented with sublethalamounts of H2O2. Moreover, the SOD inhibitor sodiumdiethyldithiocarbamate blocks PCD progression in thesecells (Delledonne et al. 2001). This proves that duringHR, NO does not require the presence of O2

2, but needsH2O2 (produced after the dismutation of O2

2 catalysedby SOD) in order to activate the PCD machinery.However, in other cases, NO and H2O2 do not seem to co-operate and only one of them is enough to cause PCD.For instance, in Arabidopsis suspension cultures, highconcentrations of NO are sufficient to induce PCD inde-pendently of H2O2 (Clarke et al. 2000).

In an intriguing new study, the map-based positionalcloning of rice NOE1, a gene whose knockout leads toan excess of NO, revealed that this is actually the ricecatalase OsCATC (Lin et al. 2011). Mutant noe1 plantsare characterized by increased leaf H2O2 concentrations,which in turn promote the synthesis of NO by the activa-tion of the enzyme NR. This demonstrates that in somescenarios NO is produced downstream of H2O2 as well.Since the removal of NO alleviates the symptoms ofcell death in the noe1 mutants, in this case NO seemsto serve as an endogenous mediator of H2O2-inducedcell death.



As described earlier, stomatal closure initiated by ABAis realized through an intracellular signalling pathwaythat includes H2O2 and Ca2+-permeable channels.However, another major component in this chain ofeffectors appears to be NO. Indeed, exogenous NO isactually able to induce stomatal closure. IncreasedH2O2 concentrations provoked by ABA may in turntrigger NO generation by NR and nitrogen oxide synthase(NOS)-like enzymes (Neill et al. 2008). This presumablyoccurs through the action of the protein kinase OXI1and involves Ca2+ (Rentel et al. 2004). Another elicitorof stomatal closure is extracellular calmodulin(ExtCaM), which plays an important physiological rolein the regulation of stomatal diurnal rhythm. Extracellu-lar calmodulin and ABA can induce some parallelchanges in second messenger levels in guard cells, in-cluding H2O2 and NO. In the ExtCaM-mediated stomatalclosure scenario, H2O2 first accumulates after activationof GPA1—the a subunit of a heterotrimeric G protein,further downstream follows the AtNOA1-dependent NOaccumulation, which ultimately leads to stomatalclosure (Li et al. 2009).

In a recent study, genetic screening for mutants de-fective in H2O2-induced NO accumulation has identifiedthe prohibitin gene PHB3 as a player in the NO homeo-stasis system (Wang et al. 2010b). Remarkably, phb3mutations do not appear to affect H2O2 metabolism orsignalling, positioning prohibitin only in the NOpathway. These findings prove that although H2O2 andNO influence and partly complement each other, theirdownstream networks diverge at some points.

Conclusions and forward lookIn the course of evolution, plants developed mechan-isms to utilize potentially toxic ROS for signalling pur-poses. This turns ROS, and mainly H2O2, into signallingagents with tremendous impact on plant growth and be-haviour. Since accumulation of higher concentrations ofH2O2 is a potent inducer of cell death, a major scientificchallenge is to reveal the regulatory processes thatgovern H2O2 metabolism and find a way to prevent ex-cessive H2O2 synthesis in the field. Although the progressmade in the last few years contributed to the overallknowledge concerning H2O2 physiology and unveiledmany components in its signalling network, our knowl-edge is only scratching the surface. Their answers willhave not only fundamental, but also practical import-ance, since crop yield depends on the capacity ofplants to tolerate elevations in ROS. Perhaps the futuredeciphering of the subtle mechanisms of plants to fine-tune the H2O2 concentration holds the key for improving

agriculture and responding to the demands of the ever-growing world population.

Sources of fundingThis work was financially supported by EC FP7, 245588project ‘Biosupport’ and by the Multidisciplinary ResearchPartnership ‘Ghent Bio-economy’.

Contributions by the authorsV.P. and F.B. wrote the paper.

AcknowledgementsWe thank Annick Bleys for help in preparing the manu-script and Viktor Ivanov for designing the figures.

Conflict of interest statementNone declared.

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