Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2005?

2005
28410121020
Original Article
Oxidative stress and ozone: perception, signalling and response
M. Baier
et al.

Plant, Cell and Environment (2005) 28, 10121020

Oxidative stress and ozone: perception,

signalling and response
MARGARETE BAIER, ANDREA KANDLBINDER, DORTJE GOLLDACK & KARL-JOSEF DIETZ

Biochemistry & Physiology of Plants, W5, University of Bielefeld, 33501 Bielefeld, Germany

ABSTRACT
The primary site of ozone interaction with plant cells is the
extracellular matrix where ozone challenges the antioxidant protection of the cells. Accordingly, ozone sensitivity
generally correlates with the ascorbate status of the apoplast, which is an important signal initiation point. In addition, ozone sensing takes place by covalent modification of
redox-sensitive components of the plasma membrane, for
example ion channels like the plasma membrane Ca2+channels. Subsequent intracellular signal transduction is an
intriguing network of hormone, Ca2+ and MAPK signalling
pathways, significantly overlapping with oxidative burstinduced pathogen signalling. Comparison of recent
transcriptome analysis revealed that in addition to genes
generally induced by all kinds of oxidative stress, for example, transcripts for PR-proteins and most antioxidant
enzymes, approximately one-third of the responsive transcripts are ozone specific, indicating jasmonic acid, salicylic
acid and ethylene-independent redox signalling triggered
by extracellular redox sensing.
Key-words: abiotic stress; oxidative stress; ozone; reactive
oxygen species; redox signalling; signal transduction;
transcriptomics.
Abbreviations: 2CPA, 2-Cys peroxiredoxin-A; ABA,
abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic
acid; GSH, reduced glutathione; JA, jasmonic acid; MAPK,
mitogen-activated protein kinase; RNS, reactive nitrogen
species; ROS, reactive oxygen species; SA, salicylic acid;
UV, ultra violet.

INTRODUCTION
Plants face a continuously changing environment. In order
to complete their life cycle and maintain a sufficient level
of fitness to reproduce, they need to adapt to abiotic and
biotic conditions. Periods of deviation from optimum are
sensed in order to anticipate severe stress and to activate
hardening processes. Two basic mechanisms allow for sensing of such environmental cues. First, sensors and receptors
have evolved to directly monitor physical or chemical
Correspondence, Margarete Baier.
E-mail: margarete.baier@uni-bielefeld.de

1012

parameters (sensors) or to catch transient chemical signals.

Alternatively, response reactions are triggered indirectly by
modified metabolic activities. One particular element of
metabolic imbalance often involved in adaptation processes is the stimulated production of reactive oxygen species (ROS) (Van Breusegem et al. 2001). ROS oxidize
cellular constituents such as lipids, proteins and nucleic
acids and can initiate radical chain reactions. An intricate
network of defence and repair mechanisms counteracts
these oxidation reactions. Imbalances between ROS generation and safe detoxification represent metabolic states that
frequently are referred to as oxidative stress. In order to
understand oxidative signalling, the chemical type of ROS
and the subcellular site of generation are of importance.
In biological systems, oxidative stress results from the
presence of elevated levels of oxidizing agents that are able
to abstract electrons from essential organic molecules and
disturb cellular functions. The most commonly encountered
oxidizing molecules in the cell are reactive oxygen species
(ROS) that derive from the abundantly available and
rather inert dioxygen (O2) (Elstner 1990). Direct transfer
of one electron to O2 produces the superoxide anion radical
(O2-) that subsequently acts as oxidant in single electron
transfer reactions to form hydrogen peroxide (H2O2),
hydroxyl radical (OH) and finally H2O. Due to its exceedingly high reactivity OH will react within diffusion distance
with any organic molecule and is unlikely to serve as a
signalling molecule whereas such function is assumed for
O2-, H2O2, O2 as well as reactive nitrogen species such as
peroxynitrite (ONOO) (De Gara, de Pinto & Tommasi
2003; Neill, Desikan & Hancock 2003; Dietz 2003).
In photosynthetic cells, excess light affects the intracellular redox homeostasis and can evoke oxidative stress similar to O3. In both cases (i) O2- and H2O2 are discussed as
signalling molecules; (ii) the signals are produced distant
from the nucleus, where transcriptional regulation takes
place and information has to be transferred across membranes; and (iii) the low molecular weight antioxidants
ascorbate and glutathione generally participate in the
regulation of the cellular redox homeostasis. The major
difference between sensing oxidative stress caused by
atmospheric O3 and intracellularly produced ROS is that in
plasmatic compartments the thiol system is employed and
could sense ROS information at the site of production (Pastori & Foyer 2002; Dietz 2005). In contrast in the secretory
pathway and the apoplast, thiols are mainly in the oxidized
2005 Blackwell Publishing Ltd

Oxidative stress and ozone: perception, signalling and response 1013

disulphide form (Vitale & Denecke 1999), which makes
direct sensing of oxidative stress by thiol/disulphide proteins impossible.
This review summarizes aspects of oxidative stress, how
they might be sensed, as well as how and which specific or
general responses are induced. The immense body of literature available on all aspects of ROS in plants thwarts any
attempt to cover this topic comprehensively. Instead, in the
following, selected and recent results of physiological
responses to oxidative stress focusing on ozone-induced
adaptive mechanisms will be reviewed.

OZONE SENSING AND PRIMARY

OZONE RESPONSES
Sensing ozone by its reaction products
At the cellular level, in leaves of ozone-sensitive plants
acute exposure to high ozone can induce chlorosis and
necrotic lesions whereas accelerated leaf senescence has
been observed for chronic exposure to low ozone levels
(Schlagnhaufer et al. 1995; Rao, Koch & Davis 2000a; Rao,
Lee & Davis 2002; Mahalingam et al. 2003). As a mechanism for these ozone-induced damages the generation of
reactive oxygen species as superoxide and hydrogen peroxide by ozone degradation in the apoplast has been proposed.
Ozone sensitivity is generally correlated with the ascorbate status of the leaf tissue (Conklin & Barth 2004), which
was genetically confirmed by isolation of ascorbic acid biosynthetic mutants on the basis of their increased sensitivity
to O3 fumigation (Conklin et al. 1999). The antioxidant
ascorbate accumulates to millimolar concentrations in leaf
apoplasts and may scavenge there significant amounts of

ozone (Noctor & Foyer 1998; Plchl et al. 2000). Accordingly, when for instance in an ozone-sensitive genotype of
Raphanus sativus L. the L-ascorbate content was increased
by supplying the biosynthetic precursor L-galactono-1,4lactone, tolerance to ozone was increased (Maddison et al.
2002). However, using quite high concentrations of
300 p.p.b. O3, Luwe, Takahama & Heber (1993) observed a
very intriguing time-dependent relationship between oxidation of extracellular ascorbate and subsequent oxidation
of the intracellular glutathione pool, while the cellular
ascorbate redox state was unaltered during fumigation.
Ascorbate regeneration was tightly coupled to GSH within
the cell and transport activity was indicated to replenish the
reduced ascorbate pool in the apoplast. By the glutathionedependent regeneration mechanism, the extracellular
ascorbate oxidation affected the intracellular thiol
signature.
Experimental work strongly indicates that ascorbate is
not the only signal initiation point in ozone sensing. As
demonstrated by Jacob & Heber (1998) using oxidationsensitive fluorescence dyes, even co-infiltration of leaves
with 10 mM ascorbic acid did not prevent ascorbic-independent oxidation reactions. The reactivity of ozone is too high
to be selective. From recent work several potential pathways of redox-dependent signalling can be deduced
(Fig. 1): Using aequorin as an intracellular Ca2+-indicator
Clayton et al. (1999) showed a specific increase in the intracellular Ca2+-concentration if atmospheric ozone reached
concentrations above 70 p.p.b. It indicates oxidative activation of Ca2+ channels similar to the response to ABAinduced H2O2 (Kwak et al. 2003; Suhita et al. 2004; Dietrich
et al. 2004). In accordance with this hypothesis, short-term
exposure to O3 decreased the stomata aperture (McAinsh
et al. 2002). In terms of signalling, the Ca2+ channel would

2+

Ca

?
Lipid
lipid signalling/JA

LOOH

O3
H2O2

H2O2

5
ascorbate

ascorbate

GSSG

dehydroascorbate

dehydroascorbate

GSH

O3

intercellular
gas space

cell
wall

plasma
membrane

ROS

cytosol

2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 10121020

Figure 1. Model of possible O3dependent signalling pathways: (1)

Oxidative activation of Ca2+-channels
triggers Ca2+ influx into the cytosol, (2) yet
unknown receptors sense oxidative stimuli
in the apoplast and transduce the
information to the cytosol, (3) enzymatic or
non-enzymatic oxidation of unsaturated
lipid to peroxides may initiate lipid-derived
signalling, (4) H2O2 generated in the
apoplast may diffuse into the cytosol, (5)
extracellularly oxidized ascorbate is
regenerated in the cytosol at the expense
of (6) glutathione; both low molecular
weight antioxidants are known to affect
metabolic activities and gene expression.
Finally, without reacting in the apoplast
ozone may diffuse across the cell
membrane, generate reactive oxygen
species (ROS) in the symplast that trigger
biochemical and genetic responses (7).

1014 M. Baier et al.

function as ROS sensor. Thereby, ROS and free radicals not
only can affect stomatal ion conductance but also ion channels from other tissues. Consequently, redox-dependent
alterations in ion conductance might be a more general
signalling pathway under oxidative stress [Fig. 1, (1)]. It is
tempting to assume that ROS interact directly with plasma
membrane bound receptors and trigger downstream events
in the cytosol. This mechanism has tentatively been
described for the animal system, where ROS activates the
epidermal growth factor receptor to undergo Tyr phosphorylation (Esposito et al. 2003) [Fig. 1, (2)]. Furthermore,
nonenzymatic or lipoxygenase-mediated break down of lipids [(3)] (Munnik & Meijer 2001), ROS, in particular H2O2
as diffusible messenger [(4)] (Laloi, Apel & Danon 2004),
modulation of cytosolic ascorbate [(5)] and glutathione
[(6)] relations, respectively (Noctor & Foyer 1998; Gomez
et al. 2004) are well established regulatory and signalling
compounds and may represent other routes of O3 triggered
signalling from the site of chemical reaction of O3 in the
apoplast or plasma membrane to the cytosol.

Oxidative burst
Recent studies presented evidence that ozone activates an
oxidative burst and may induce a hypersensitive response
(HR) similar to defence responses of plants to pathogen
infections (see Kangasjrvi et al. 2005 for details). Involvement of the plasmamembrane NADPH oxidase in ozonetriggered ROS accumulation has been, for example, shown
in the Arabidopsis thaliana mutant rcd1 (radical-induced
cell death 1; Overmyer et al. 2000). In this work, application
of the NADPH oxidase inhibitor diphenylene iodonium
inhibited ROS accumulation and reduced leaf damage of
the rcd1 mutant indicating that ozone initiates active cellular ROS production (Overmyer et al. 2000). In addition, in

ROS

Cytosol
C a 2+

Nucleus

MAPK

Ethylene

SA

JA

PP2C

Figure 2. Control of protein phosphorylation cascades. Oxidative

2+

bursts induce Ca -efflux, which activates protein phosphorylation.

MAPK induce ethylene production, which stimulates MAPKsignalling. SA promotes ethylene, while JA inhibits SA-induction
of ethylene biosynthesis. In feed-forward loops MAPKs induce
expression of signalling kinases, which than increase the
phosphorylation activity. Antagonists are phosphatases like PP2C,
which can be redox-regulated themselves.

leaves of tobacco cultivar Bel W3 Pasqualini et al. (2003)

observed an ozone-induced oxidative burst accompanied
by transient increase of transcript levels of the hypersensitive response marker pathogenesis-related-1a. The tobacco
leaves displayed increased protease activity and chromatin
condensation indicating that ROS-induced programmed
cell death is initiated by ozone-induced oxidative stress
(Pasqualini et al. 2003). As another example, in transgenic
tobacco plants with reduced catalase activity an active cell
death programme was triggered by changes in H2O2
homeostasis and involvement of a signalling cascade leading to an NADPH oxidase-dependent burst has been suggested (Dat et al. 2003).

INTRACELLULAR SIGNAL TRANSDUCTION

Downstream oxidative signalling involved in mediating the
ozone responses within the cell is a two-step response process comprised of (i) fast-signalling events including a rapid
change in the Ca2+ signature and protein phosphorylation,
and (ii) the activation of auxiliary signalling pathways,
which stabilize and amplify the primary signal (Fig. 2).

Oxidatively induced Ca2+-signals rapidly activate

protein phosphorylation
Ca2+-release by oxidative activation of redox-sensitive Ca2+channels (Clayton et al. 1999; McAinsh et al. 2002) causes
rapid changes of the protein phosphorylation pattern
(Agrawal, Rakwal & Iwahashi 2002a, Agrawal et al. 2002b)
(Fig. 2). Signal duration is controlled by Ca2+-pumps, for
example, in the ER-membrane (Sze et al. 2000) and induction of Ca2+-binding proteins (Agrawal et al. 2002c).
Mitogen-activated protein kinase (MAPK)-cascades
play key roles in the change of the phosphorylation pattern
(Samuel, Miles & Ellis 2000; Morris 2001). For example in
rice seedlings a 66-kDa ERK-type MAPK is one of the
earliest phosphorylated proteins. Ozone-induced phosphorylation stabilizes the enzyme (Agrawal et al. 2002b), which
increases the kinase activity. Studies with the protein phosphatase inhibitors cantharidin, endothall and okadaic acid
revealed that in addition transcription, as, for example, of
the MAPK OsBMWK1, is stimulated by protein phosphorylation (Agrawal et al. 2003a). The MAPK-signal is controlled by redox-sensitive protein phosphatases 2C (PP2C),
like the alpha-alpha PP2C MP2C, which is induced under
oxidative conditions and regulates signal transduction specificity by supporting SIMK signalling via specific inhibition
of the kinase SAMK (Meskiene et al. 2003).

Phytohormones control the signal

The plant hormones jasmonic acid (JA), salicylic acid (SA),
ethylene and ABA control the ozone response. Thereby, in
ABA-signal transduction the ABA-antagonistic PP2Cs
ABI1 and ABI2 are inhibited by H2O2 (Meinhard & Grill
2001; Meinhard, Rodriguez & Grill 2002). Therefore, under
oxidative stress the MAPK-mediated ABA signal is

2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 10121020

Oxidative stress and ozone: perception, signalling and response 1015

strengthened. With ABA-signalling leading to H2O2 generation (Jiang et al. 2003), the redox-sensitivity of the PP2C
exponentially amplifies the primary signal. In this context
it should be noted that several ABA responsive genes were
activated in the Arabidopsis mutant vtc1 (Pastori et al.
2003), which has only 30% of wild-type ascorbate. This
observation indicates that the threshold of the redox regulation of ABA-signal induction is low. A putative initiation
site for ABA signal induction is the violaxanthin cycle. It is
activated, if photosynthetic electron transport demands for
energy dissipation (Demmig-Adams & Adams 1996). Low
availability of reduced ascorbate arrests it in the epoxide
status (Neubauer & Yamamoto 1994) supporting biosynthesis of the ABA precursor xanthoxin (Marin et al. 1996).
Regulation of the induction and spreading of oxidative
stress symptoms by the phytohormones ethylene, SA and
JA has been reported in several recent studies (Rao et al.
2002; Vahala et al. 2003; Moeder et al. 2002). In the Arabidopsis mutant rcd1, for instance, exogenous ethylene promoted superoxide-dependent cell death whereas
exogenous application of methyl jasmonate inhibited its
propagation. However, jasmonates, as well as ethylene, are
involved in lesion containment (Overmyer et al. 2000).
Based on studies of the O3-sensitive, JA-insensitive Arabidopsis mutant jar1 and the O3-tolerant, ethylene-insensitive
ein2 mutant, Tuominen et al. (2004) hypothesized that early
accumulation of ethylene stimulates spreading of cell death
and suppresses protection by JA. Late accumulation of JA,
however, inhibits the ethylene pathway and the propagation of cell death (Tuominen et al. 2004).
JA, SA and ethylene are second messengers in oxidative
signal transduction. These phytohormones can mimic O3
(Agrawal et al. 2003b) and are generally induced by oxidative bursts (Watanabe, Seo & Saki 2001). Jasmonic acid,
salicylic acid and ethylene regulate the strength of the primary signal by triggering MAPK cascades (Fig. 2; Agrawal
et al. 2003a, b; Jiang et al. 2003; Guo & Ecker 2004). In
general, ethylene and SA amplify the oxidative signal,
while JA constricts ozone-induced damage (Watanabe et al.
2001; Langebartels et al. 2002; Kangasjrvi et al. 2005).
Physiological analysis of the Arabidopsis mutant oji1
(ozone-sensitive and jasmonate-insensitive) (Kanna et al.
2003) demonstrated that JA signalling antagonizes ozoneinduced induction of ACC synthase-catalysed ethylene biosynthesis (Bae et al. 1996) and analysis of NahG and npr1
plants, which are impaired in SA signalling, revealed that
ozone-induced ethylene production depends on SA (Rao
et al. 2002).
Biosynthesis of JA and SA are controlled by plastid lipid
metabolism (Kachroo et al. 2003), lipid peroxidation and by
oxidative inactivation of antioxidant enzymes like ascorbate peroxidases and peroxiredoxins (Mano et al. 2001;
Knig et al. 2002). In parallel photosynthetic imbalances
stimulate energy dissipation processes such as the violaxanthin cycle, which forms the precursors of ABA biosynthesis (Marin et al. 1996). It can be suggested that in
oxidative signalling chloroplast metabolism, and photosynthesis in particular, control the signal strength and the

ROS
JA and SA

ABA
PP2C

MKK1

Ethylene

MPK4

Response
A

MKK4/5

MPK6

MPK3

Response
B

MEK1/2

Response
C

Figure 3. Transmission of oxidative signal takes place via at least

three MAPK signalling cascades. MPK3, MPK4 and MPK6 are
generally stress induced, while MPK1 and MPK2 mediate the ABA
response which is redox controlled by ROS activation of
antagonistic PP2C and by MPK3. MPK6 action, which transmits
the ethylene signal, inhibits MPK4 activation, while MPK3
stimulates ABA-signalling.

integration of environmental parameters not only by

photo-oxidative ROS generation (Baier & Dietz 1999;
Chang et al. 2004), but also by biosynthesis of second messengers. Therefore, even for O3, which attacks plant cells
from the outside, within the plant cell general oxidative
signal transduction pathways including chloroplast-tonucleus signalling are likely to be involved in the regulation
of plant responses.

Complexity, redundancy and specificity in

oxidative signal transduction
In the transmission of secondary signals like JA, SA, ethylene and ABA MAPK are involved. Arabidopsis thaliana
encodes 10 MAPKKKK, 80 MAPKKK, 10 MAPKK and
23 MAPK (Jonak et al. 2002), which form complex signalling networks with synergistic and antagonistic links
(Fig. 3). The double jeopardy of MAPK-signalling was
described in tobacco lines over-expressing or suppressing
salicylate-induced protein kinase (SIPK), as both manipulations led to higher ozone sensitivity (Samuel & Ellis
2002). SIPK, like the wound induced MAPK WIPK (Seo,
Sano & Ohashi 1999), is activated by the MAPKK MEK2
(Yang, Liu & Zhang 2001). During ozone exposure, activation of SIPK was prolonged in over-expressing lines, while
activation of WIPK occurred in SIPK-suppressed lines.
In Arabidopsis thaliana, the MAP kinases MPK3, MPK4
and MPK6 are activated by various abiotic stresses
(Ichimura et al. 2000; Kovtun et al. 2000) and might be central elements of oxidative signal transduction (Fig. 3).
MPK6 and MPK3, which are the Arabidopsis homologues
of SIPK and WIPK, respectively, are activated by the MAP
kinase kinases MKK4 and MKK5 (Asai et al. 2002) and the

2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 10121020

1016 M. Baier et al.

MAPKKK ANP1 (Kovtun et al. 2000). In MPK6-silenced
plants MPK4 is activated (Menke et al. 2004) indicating that
MPK6 suppresses MPK4 activation. As MPK4 is under
control of MKK1 (Huang et al. 2000), Menke et al. (2004)
suggested that in the response to wounding two MAPK
cascades act in parallel. In addition, SA-independent
MAPK-regulated signal transduction takes place. For
example, Jiang et al. (2003) showed recently that the SAinsensitive ABA-MAPK is mediated by the MAPKK
MEK1/2. MPK3 over-expressing Arabidopsis are more sensitive to ABA during germination (Lu et al. 2002), demonstrating that there is cross-talk between the MPK3- and
ABA-signalling cascades. Ethylene signalling, which is
often antagonistic to ABA-signalling (Ghassemian et al.
2000), is transmitted through the SIMK-homologue MPK6
(Guo & Ecker 2004).
The complexity and dynamics of MAPK-regulated oxidative signalling becomes obvious by comparison of promoter regulation of the cytosolic ascorbate peroxidase
APx2 and the nuclear encoded chloroplast 2-Cys peroxiredoxin 2CPA. The promoters of the two peroxidases both
respond to ABA and to redox signals (Fryer et al. 2003;
Baier, Strher & Dietz 2004). APx2 is synergistically
induced by ABA and oxidative stress (Fryer et al. 2003),
while 2CPA is oxidatively induced by wounding and photooxidative stress via one MAPKK, but strongly suppressed
by ABA via an antagonistically responding MAPKK
(Baier et al. 2004). Like with 2CPA regulation, the wounding response of Apx2 cannot be mimicked by JA (Baier
et al. 2004; Chang et al. 2004), which indicates transmission
by other secondary messengers with a specific impact of
ABA.

GENE TARGETS OF OXIDATIVE SIGNALLING

IN RESPONSE TO OXIDATIVE STRESS
AND OZONE
New insights into the responses to oxidative stress signalling and the specificity of secondary messengers have been
provided recently by PCR-based suppression subtractive
hybridization (Mahalingam et al. 2003), transcriptome analysis investigating responses to oxidative stress in wild-type
plants (Desikan et al. 2001; Tamaoki et al. 2003a) and the
analysis of transgenic plants and mutants with various
genetic backgrounds (Vranova et al. 2002; Tamaoki et al.
2003b). Based on comparison of transcriptome patterns in
plants treated with O3 (Tamaoki et al. 2003a), H2O2, sublethal doses of methyl viologen (Vranova et al. 2002) or
pathogens (Durrant et al. 2000; Maleck et al. 2000), it has
been estimated that oxidative stress affects approximately
2% of the plant transcriptome (Tamaoki et al. 2003a).
Characterizing the transcriptional responses of Arabidopsis to, for example, ozone and to pathogen treatment
Mahalingam et al. (2003) distinguished between shared
responses of ozone and pathogen exposure and transcripts
specifically regulated by ozone. By hybridization of Arabidopsis-cDNA-macroarrays Tamaoki et al. (2003a) identified 205 ozone-responsive transcripts after 12 h exposure to

O3 (200 nL L-1) and comprehensively compared the signalling pathways of ethylene, JA and SA on ozone-responsive
gene expression. Approximately 75% of the ozoneresponsive transcripts were induced and 48 of 205 genes
were suppressed by O3. Among the 109 transcripts with
known functions, 33 are involved in metabolism like monodehydroascorbate reductase, glutaredoxin and pyruvate
kinase, 24 in cellular organization and biogenesis, 25 in cell
rescue/defence (e.g. glutathione S-transferase, PR4, Lascorbate peroxidase), 11 in signal transduction like cyclophilin ROC7, six in energy (e.g. chlorophyll a/b-binding
protein and the gamma subunit of the mitochondrial F1ATPase), five in protein synthesis and degradation, three
in transcription and two in transport. This study corresponds
to results of previous studies of genes for which activation
or suppression by O3 has been described. In response to O3
exposure, transcript levels of cytosolic ascorbate peroxidase
(Willekens et al. 1994; Kubo et al. 1995), lipoxygenase
(Maccarrone, Veldink & Vliegenhardt 1992), blue copperbinding protein (Langebartels et al. 2000), glutathione Stransferase, PR proteins and catalases (Lim, Woo & Nam
2003) were elevated, whereas transcript levels of chlorophyll a/b-binding protein (Miller, Arteca & Pell 1999), rbcS
(Glick et al. 1995; Miller et al. 1999), and Cu/Zn superoxide
dismutase (Kliebenstein, Monde & Last 1998) were
reduced. Additionally, elevated transcript levels of enzymes
such as glutathione S-transferase, glutaredoxin, cyclophilin
ROC7 and ascorbate peroxidase indicate the involvement
of thiol-/disulphide transitions and thiol-dependent redox
regulation in acclimation to O3 exposure. With a proteomics
approach Agrawal et al. (2002c) studied the effects of ozone
exposure in rice. The ozone treatment resulted, for example, in decreased accumulation of photosynthetic proteins
whereas induced accumulation was, for example, observed
for an ascorbate peroxidase, superoxide dismutase, and
pathogenesis-related (PR) proteins. By contrast to acute
treatments, chronic exposure to ozone in the field caused a
smaller proportion of the Arabidopsis thaliana transcriptome to be altered, with five times more genes downregulated than up-regulated (Miyazaki et al. 2004).
As exposure to high levels of O3 induces the synthesis of
ethylene, JA and SA and activates the corresponding signalling pathways (Sharma & Davis 1997; Rao et al. 2000a,
b; Kangasjrvi et al. 2005), the regulation of 157 O3-induced
genes was analysed in signal transduction deficient mutants
as the ethylene-insensitive ein2, JA-resistant jar1 and SAinsensitive npr1. While approximately 50% of the transcripts were inhibited in ein2 and jar1, only 20% of the
transcripts were inhibited in npr1 (Tamaoki et al. 2003a),
indicating that JA and ethylene signalling are more crucial
for the induction than SA signalling at least under these
particular conditions. Of the O3-induced genes with function in cell rescue and defence such as L-ascorbate peroxidase, glutathione S-transferase and hevein-like protein
(PR4) about 70% were controlled by ethylene and JA and
were suppressed by SA, suggesting that ethylene and JA
signalling are important for the induction of defence gene
expression as outlined above.

2005 Blackwell Publishing Ltd, Plant, Cell and Environment, 28, 10121020

Oxidative stress and ozone: perception, signalling and response 1017

In the study presented by Tamaoki et al. (2003a) 16 genes
were identified whose expression was induced by all three
signalling molecules. However, about 30% of 157 induced
O3-responsive transcripts were independent of ethylene, JA
and SA pathways indicating control by other still unknown
factors. By the comparison of 205 ozone-responsive transcripts in response to drought, salinity, UV-B, low temperature, high temperature and acid rain as ROS (Tamaoki
et al. 2004) found that the transcriptional responses to O3
and UV-B stress are similar, but distinct from the responses
to the other stresses. It may be concluded that O3 and UVB share common features via the generation of reactive
oxygen species (Mackerness et al. 1999). The comparison of
the transcript regulation patterns has provided important
clues to the specificity of oxidative stress responses and
allows differentiating O3 induced responses from regulation
in context of photosynthesis and other sources of ROS.
Detailed functional characterization of transcripts and proteins identified in transcriptomic and proteomic studies will
provide further information on physiological responses to
oxidative stress in plants.
In plant cells, scavenging of reactive oxygen species is
mediated by antioxidant metabolites, for example, as ascorbate, glutathione, and tocopherols as well as by detoxifying
enzymes, for example, as superoxide dismutase, ascorbate
peroxidase, peroxiredoxin and catalase (reviewed in Mittler 2002; Neill, Desikan & Hancock 2002; Dietz 2003).
Recently, in a transgenic approach, increased tolerance to
oxidative stress that correlated with decreased membrane
damage has been shown in tobacco over-expressing
Chlamydomonas glutathione peroxidase (Yoshimura et al.
2004). As another mechanism for intracellular ROSscavenging accumulation of the antioxidant thylakoid
membrane-bound polyamine putrescine was induced by
enhanced ozone concentrations in ozone-tolerant, but not
in ozone-sensitive tobacco (Navakoudis et al. 2002). Consequently, a regulatory role of polyamines for the adaptation
of the photosynthetic apparatus has been suggested (Navakoudis et al. 2002). Another antioxidant compound isoprene has a role in quenching H2O2 and decreased lipid
peroxidation of membranes (Loreto et al. 2001; Loreto &
Velikova 2001).

OUTLOOK
Recent analysis of transcriptome patterns in wild-type
plants and mutants has expanded our knowledge about
responses that are induced by oxidative stress. However, in
contrast to the experimental approaches, where, for example, the ozone concentration was suddenly and strongly
increased, in nature, during a vegetation period, the maximum daily ozone concentrations peak several times a
month over periods of weeks and induce hardening of the
plants (Luwe 1996). Therefore, one can assume that most
responses described in short-term fumigation experiments
are acute stress reactions. To understand oxidative signalling in general, however, we will have to distinguish the
intracellular oxidative sensing mechanisms from the com-

plex networks observed in short-term stress experiments.

Defining the switch board of oxidative signalling and the
thresholds of particular signalling pathways will challenge
us in the coming years. Besides introducing more and more
defined transcriptomic approaches, analysis of promoter
responses in physiological contexts may guide us to the
cellular redox signalling networks. In this context, recently
investigated examples are the analysis of the APx2, PetE1
and 2CPA promoters in Arabidopsis thaliana (Oswald et al.
2001; Fryer et al. 2003; Chang et al. 2004; Baier et al. 2004).
Thereby, the APx2 promoter responds to photosynthetically produced ROS (Chang et al. 2004), the PetE1 promoter is controlled by the redox state of the plastoquinone
pool as long as the concentration of sugars is low (Oswald
et al. 2001), and the activity of the 2CPA promoter correlates, like that of the thioredoxin redox regulation system
(Schrmann & Jacquot 2000), with the electron pressure on
ferredoxin and the redox state of the [NADP+ + NADPH]
pool (Baier et al. 2004). While forward genetic screens for
mutants affected in PetE1 promoter regulation led to isolation of mutants defect in the transmission of the plant
hormone abscisic acid (ABA) (Huijser et al. 2000), physiological analysis of reporter gene plants showed that ABA,
which can cause oxidative bursts (Kwak et al. 2003) and
whose transmission is redox regulated (Meinhard & Grill
2001; Meinhard et al. 2002), is a modulator of redox-active
promoters like that of APx2 (Fryer et al. 2003) and 2CPA
(Baier et al. 2004). Constitutive induction of APx2 promoter activity in rax1 (Ball et al. 2004), demonstrates that
glutathione-regulated thiol switches can play an important
role in transmission of ROS signals. Future work such as
the analysis of the rimb-mutants, which were screened for
lower 2CPA promoter activity and show impairment of
several redox-regulated genes relative to the redox state of
the ascorbate pool (Heiber et al. 2004), will point to further
regulators of intracellular redox signalling networks which
adjust nuclear gene expression to physiological variation of
the redox status.

ACKNOWLEDGMENTS
The authors work that is summarized in this review was
supported by the Deutsche Forschungsgemeinschaft
(FOR387, Di346/6; Ba 2011/2)

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for publication 6 January 2005

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