1. Tropospheric ozone as a fungal elicitor 1
J. Biosci. 34(1), March 2009
1. Introduction
Tropospheric ozone (O3
) is recognized as the most phytotoxic
among the common air pollutants (Sandermann et al 1998),
and is responsible for a wide range of damages to plants. The
chemical characteristics at the basis of its behaviour are the
high oxidizing power, a diffusion coefficient similar to the
one of CO2
(and consequently a certain facility to penetrate
plant tissues), solubility in water 10 times higher than CO2
and tendency to react with water in a sub-basic environment
(Izuta 2006).
Its noxious activity towards plants can occur in both
direct (e.g. through liberation of hydroperoxides) and indirect
ways (e.g. through liberation of hydroximetylperoxides);
these aspects will be discussed in detail in the following
sections.
O3
is formed in the troposphere by the energy released
from electrical discharges, or can go down from the
stratosphere; nevertheless, it is mainly generated through
the photolytic cycle of O3
, a series of chemical reactions
triggered by hydrocarbons and nitrogen oxides present in
exhaust gases from vehicles (Crutzen 1973; Chameides
and Lodge 1992). The cycle is started by the hydroxylic
radical, a highly reactive molecule formed when a radical
oxygen, generated spontaneously in the stratosphere by
splitting of O3
, reaches the troposphere and there reacts with
H2
O. At the same time, the hydroxylic radical can oxidize
anthropogenic pollutants to smaller chemical specimens that
are more easily eliminated. The synthesis or degradation of
O3
depends on the NO2
/NO ratio: the higher the ratio, the
higher the O3
, and vice versa.
O3
concentrations in the troposphere regularly exceed
national and international limits in Europe and North
America (Hough and Derwent 1990; Flatøy et al 1996),
ranging typically between 20 and 60 nl l-1
with peaks of up
to 250 nl l-1
(Stockwell et al 1997), and some models predict
a further increase of 0.3–1% per year over the next 50 years
(Liao et al 2006).
Under the stimulus of the environmental problems
connected with tropospheric O3
, many researchers have
focused, during the past decades, on the study of its effects
on plants, and several proprieties that can be utilized for
convenient practical application have been brought to light
(Eckey-Kaltenbach et al 1994; Sudhakar et al 2006).
http://www.ias.ac.in/jbiosci J. Biosci. 34(1), March 2009, 000–000, © Indian Academy of Sciences 000
Review
Tropospheric ozone as a fungal elicitor
PAOLO ZUCCARINI
Department of Crop Biology, – Section of Plant Physiology, University of Pisa, Pisa, Italy
(Fax, 0039 050 2216532; Email, p.zuccarini@virgilio.it)
Tropospheric ozone has been proven to trigger biochemical plant responses that are similar to the ones induced by
an attack of fungal pathogens, i.e. it resembles fungal elicitors. This suggests that ozone can represent a valid tool
for the study of stress responses and induction of resistance to pathogens. This review provides an overview of the
implications of such a phenomenon for basic and applied research. After an introduction about the environmental
implications of tropospheric ozone and plant responses to biotic stresses, the biochemistry of ozone stress is analysed,
pointing out its similarities with plant responses to pathogens and its possible applications.
[Zuccarini P 2009 Tropospheric ozone as a fungal elicitor; J. Biosci. 34 000–000]
Keywords. Elicitor; ozone; pathogens; stress
Abbreviations used: ACC, 1-aminocyclopropyl-1-carboxylic acid;AOX, alternate oxidase;ATP, adenosine triphosphate; GRAS, generally
considered as safe; HR, hypersensitive response; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide
phosphate; NO, nitric oxide; NOS, NO synthase; PR proteins, proteins related to pathogenesis; PS, photosystem; ROS, reactive oxygen
species; SA, salicylic acid; SAR, systemically acquired resistance; SMV, Soybean mosaic virus; US, ultrasound; UV, ultraviolet

2. Paolo Zuccarini2
J. Biosci. 34(1), March 2009
This review focuses on the capacity of tropospheric O3
to trigger the same plant reactions as fungal pathogens, and
on the possible implications of these for agronomy and plant
pathology. First of all, the reactions that plants mount as a
consequence of biotic stresses are discussed. Subsequently,
the damaging effects caused by O3
stress on the vegetable
organism are analysed. Finally, a comparison between the
two kinds of phenomena is performed, in order to highlight
the activity of O3
as a fungal elicitor, and its possible
practical applications.
2. Plant responses to biotic stresses
When the interaction between a pathogen and its host is
species-specific, the host expresses genes of resistance with
biochemical affinity to the genes of avirulence brought by
the pathogen (the elicitors). When elicitors are recognized by
the proper receptors of the plant, they trigger the activation
of its genes of resistance, and defences are put into action
(Hahn 1996; Montesano et al 2003).
Elicitors present a wide range of structures, and can be
carried by the pathogen (exogenous elicitors) or produced by
the plant as a consequence of the plant–pathogen interaction
(endogenous elicitors); in both cases, their role is to stimulate
the defence reaction of the plant (Ebel and Cosio 1994). The
most frequent biochemical defence responses are fast death
of those cells that are directly in contact with the pathogen
(known as hypersensitive response [HR]); synthesis and
accumulation of phytoalexins and secondary metabolites;
systemically acquired resistance (SAR); synthesis of
proteins related to the pathogenic event (PR proteins).
2.1 Hypersensitive response
HR is often the main mode of resistance in the case of fungal
or bacterial attack, and is characterized by the formation of
necrotic lesions in the zones involved and limitation of growth
and spread of the pathogen.
HR is mediated by reactive oxygen species (ROS) such
as superoxide, hydrogen peroxide and the hydroxyl radical
(Watanabe and Lam 2006). These activated species produced
by the plant undergoing an attack (Apel and Hirt 2004) can
directly kill the pathogens, strengthen the cellular walls by
deposition of structural compounds such as lignin, produce
specific structural or elicitor proteins or destroy the host cell
(Baker and Orlandi 1995; Lamb and Dixon 1997).
The destruction of host cells is properly called a
hypersensitive response, and is caused by the peroxidation of
membrane lipids and loss of electrolytes. Cell ion imbalance
and the subsequent breakdown of cellular components result
in death of the affected cells and formation of local lesions.
In this way, the plant “sacrifies” parts of its tissues, but
isolates the area of action of the pathogen and prevents it
from spreading through the whole organism (Heath 2000).
The reinforcement of cell walls surrounding the
infection is intended to create a physical barrier to inhibit
the spread of infection (Pontier et al 1998); this happens
through ROS-triggered deposition of callose and oxidized
derivates of precursors of lignin, as well as by production
of hydroxyproline-rich glycoproteins (Matthews 2007).
Specific proteins produced in connection with HR can have
a structural or an elicitor role.
The role of ROS in pathogen killing and host cell
destruction has been widely demonstrated and accepted,
but evidence exists that they do not act alone (Dangl 1998).
Nitric oxide (NO) and NO synthase (NOS) play an important
role in plant defence reactions against pathogens, together
with ROS. Tobacco plants infected with a tobacco-specific
virus showed an enhancement of NOS activity (Durner et
al 1998), and similar results were observed in soybean cells
and Arabidopsis thaliana in response to a bacterial pathogen
or a proper elicitor (Delledonne et al 1998), suggesting that
NO significantly contributes to the actions performed by
ROS in the early stages of plant defence responses.
HR response is usually the first defence of the plant
against pathogenic attack; when it is not sufficient to stop the
aggression, synthesis of phytoalexins comes to the rescue.
2.2 Synthesis of phytoalexins
Phytoalexinsareagroupofphenylpropanoidswhosesynthesis
is induced by various kinds of stress; phenylpropanes
come from deamination of the amino acids phenylalanine
and tyrosine produced in the biosynthetic pathway of
shikimic acid (Hammerschmidt 1999). About 350 different
phytoalexins are known today, all coming from the same
common metabolic pathway; plants belonging to different
botanical families synthesize specific phytoalexins (Mert-
Türk 2002). Phytoalexins often do not come from synthesis
ex novo, but from the biotransformation of previously
existing molecules, e.g. by conjugation, compartmentation,
release of conjugated forms (Fuchs et al 1983; Pedras et
al 2000); this means that bigger stores of biotransformable
substances represent higher potential resistance for a plant.
Phytoalexins perform their antibiotic activity through
the destruction of pathogenic membranes, particularly the
plasmatic ones; this action is stronger for higher levels of
lipophilicity, hydroxylation and acidity of the molecule
(Cowan 1999; Ishida 2005).
2.3 Systemically acquired resistance
This phenomenon occurs when pathogen attack on a
certain part of the plant causes induction of resistance

3. Tropospheric ozone as a fungal elicitor 3
J. Biosci. 34(1), March 2009
in areas that have not been directly infected (Ryals et al
1996). The activation of systemically acquired resistance
(SAR) is constituted by a modulation or strengthening
of specific mechanisms of resistance that cause the
plant–pathogen interaction to be incompatible. However,
SAR cannot provide complete resistance from the attack
of pathogens, since for each plant–elicitor combination a
particular induction of resistance, with its own spectrum, is
generated.
SAR is divided in two phases: start (the transitory
phase comprising all the events leading to resistance) and
maintenance (semi-stationary state of resistance coming
after the initial part).
Salicylic acid (SA) plays a central role in the signal
transmission for induction of SAR; in several species, a
correlation has been demonstrated between concentration
of SA and increased resistance to biotic stresses (Mur et al
1996; Mauch-Mani and Métrauxs 1998; Mètrauxs 2001). SA
is produced in the infected tissues and transferred through
the phloem to non-infected ones, where it induces resistance,
acting as a signal molecule. For induction of resistance,
SA is not only synthesized ex novo at the moment of the
infection, but plants also have supplies of its conjugated
form with glucose, which can be released when necessary.
The accumulation of these conjugated forms represents a
constitutional defence for the plant.
Other molecules capable of conferring SAR are 2,6-
dichloroisonicotinic acid and its methyl ester (Vernooj et
al 1995); the S-methyl ester of benzo (1,2,3) thiadiazol-7-
carboxylic acid (Kunz et al 1997); jasmonate and its methyl
ester (Repka et al 2004).
2.4 Proteins related to pathogenesis
The contribution of proteins related to pathogenesis (PR
proteins) to disease resistance is highly variable, and depends
both on the plant and on the pathogen (Bowles 1990).
PR proteins accumulate in hostile environments as
vacuoles in the cell walls and intercellular spaces, since their
physicochemical proprieties allow them to resist low pH and
proteolytic scission (Datta and Muthukrishnan 1999). Their
basic role is to limit the access of pathogens and to induce
programmed cell death. The types and roles of PR proteins
are discussed further in section 4.
3. Biochemistry of ozone stress
When the plant is attacked by O3
, it puts into action a
series of metabolic responses that can result in either
induction of resistance or damage. Damage can be either
acute or chronic. In this section, damage is analysed, with
special regard to the chemical specimens that activate
responses connected to it.
3.1 Acute damage
Acute damage can be defined as damage subsequent to acute
exposure of an organism to a biotic or abiotic stressor. The
exposure is acute when it lasts for a short period of time,
during which the organism undergoes severely intense
stress. In the case of an abiotic stressor such as O3
, an acute
exposure is defined as exposure to a high concentration of O3
for a short interval which is not repeated later; an example
of acute exposure could be 250 nl l-1
for 5 h (Pasqualini
et al 2007). Complexively, the three factors that interact
to distinguish between acute and chronic damage are the
severity and duration of the stress, and sensitivity of the
attacked organism.
Acute and chronic damage therefore have different
dynamics, and usually lead to different consequences.
While chronic exposure can provide the organism with
an increase in tolerance and resistance, in acute exposure
the modifications caused to the metabolism of the host are
represented most of the time by the damage itself, which
in extreme cases can lead to the death of the organism,
or to partial damage of the attacked tissues. This second
eventuality, in plants, often results in foliar necroses, and
can be a valid strategy to confine the noxious agent and
prevent it from spreading to the rest of the organism. There
are also cases in which acute O3
exposure can provide the
plant with better resistance to further pathogenic attacks,
through induced metabolic changes that can persist for days,
weeks or months (Sandermann 2000). Puckette et al (2007)
suggest that acute O3
fumigation (300 nmol mol-1
for 6 h)
on Medicago truncatula can be a valid tool to improve the
plant’s resistance to a variety of abiotic stressors, in spite of
the damages (mainly at foliar level) caused by the treatment.
For this reason, varieties with high tolerance to acute O3
represent the ideal target for this kind of treatment. Soybean
plants exposed to acute O3
fumigation while being infected
with Soybean mosaic virus (SMV) showed acquisition of
non-specific resistance against the virus that is concretized
by a significant slowing down of systemic infection and
disease development, by means of increased transcription
of fungal, bacterial and viral defence-related genes (Bilgin
et al 2008).
Acute damage is therefore the kind of damage that
generally, but not always, leads to cell death. Commonly
visible symptoms are foliar necroses, which can appear
within 15–72 h from a single acute dose of O3
(Wohlgemuth
et al2002; Neufeld et al 2006). These foliar necroses appear
similar to cases of programmed cell death subsequent to
pathogenic attacks, described by Kombrink and Somssich
(1995).
The necrotic lesions associated with acute damage
can either be caused by direct action of O3
, leading to
oxidation of cellular components and uncontrolled cell

4. death (Pell et al 1997), or by programmed cell death
triggered by the ROS produced by primary reactions
(Greenberg et al 1994; Overmyer et al 2005), depending on
the O3
concentration. A high concentration of O3
causes a
rapid attack on the cell walls and membranes, with loss of
semipermeability, plasmolysis and death. If the event is fast
and extensive, the quick and massive liberation of enzymes
from the tonoplast could lead to uncontrolled proteolysis and
uncontrolled cell death (Heath 1987b; Fiscus et al 2005).
Lower O3
concentrations can lead to a slow degradation of
the plasmatic membrane (24–48 h). This happens through
ATP-ase inhibition (Dominy and Heath 1985), alteration of
Ca2+
transport (Castillo and Heath 1990) and oxygenation of
the membrane lipids (Ranieri et al 1996).
The key molecules in acute damage are ROS and
products of lipid peroxidation which, in cases of O3
stress,
seem to act both as messengers of stress signals (induction
of activity of detoxifying systems) (Puckette et al 2007) and
as being directly responsible for the development of necrotic
lesions (Mehdy et al 1996; Langebartels et al 2002). The
fact that the same molecule can play both these roles at the
same time cannot be ruled out, and the effects can depend
on its localization in the plant. For example, tobacco
plants modified for the overexpression of mitochondrial
alternate oxidase (AOX), characterized for this reason
by lower mitochondrial ROS concentration, surprisingly
showed higher sensitivity to O3
fumigation, and a possible
explanation is that ROS-scavenging systems were activated
by the altered defensive mitochondrial-to-nucleus signalling
pathway (Pasqualini et al 2007).
Products of lipid peroxidation are generated by the action
of ROS on the polyunsaturated fatty acids of the membrane
lipids (Takamura and Gardner 1996); their direct impact on
plant metabolism involves a series of reactions that take place
at the level of chloroplasts and their antioxidant systems
(Kraus et al 1995; Mano et al 2001) and at mitochondrial
level, where the glycine decarboxilase complex is a major
target (Taylor et al 2002).
In acute damage, O3
enters the plant through the
stomata, diffuses in the apoplast and, once there, is rapidly
decomposed to hydroxylic radical, superoxide, hydrogen
peroxide and other ROS (Heath and Taylor 1997). Formation
of the hydroxylic radical is stimulated by the presence
of Fe2+
, amines, thiolic groups, caffeic acid (Grimes et al
1983; Byovet et al 1995); hydrogen peroxide is produced
by the reaction of O3
with unsaturated fatty acids (Pryor and
Church 1991).
These ROS can be detoxified by antioxidant substances
present in the apoplast and in plant mitochondria (Møller
2001); otherwise, they attack the proteins and lipids of
the plasmatic membrane through the process of lipid
peroxidation (Schraudner et al 1997). Lipid peroxide
radicals trigger various chain reactions, and the increased
level of conjugation of fatty acids reduces the elasticity
and fluidity of the membrane, creating eccentricities
inside (Heath 1987a). Lipid peroxides can be detoxified
enzymatically by means of hydrolysis operated by
phospholipases (Chandra et al 1996; Schraudner et al 1997),
while glutathione-S-transferase and glutathione peroxidase
in the cytosol detoxify the secondary products of lipid
peroxidation (Willekens et al 1994; Conklin and Last 1995);
their activation is very fast after O3
treatment. Studies on the
biphasic production of ROS in O3
-treated plants, moreover,
provide evidence for the contribution of plant endogenous
H2
O2
in the development of necrotic lesions connected to O3
stress (Schraudner et al 1998; Castagna et al 2005).
Some authors also theorize that O3
can cause acute
damage through interaction with volatile molecules
produced by the plant, which are concentrated in the
apoplast, such as ethylene, isoprene and α-pinene (Hewitt
et al 1990). Wellburn and Wellburn (1996) showed that
O3
-sensitive plants had a higher ethylene production than
the average, while tolerant plants tended to accumulate
antioxidants such as polyamines, polyphenols, ascorbate
reductase and glutathione reductase. Other research, on the
other hand, provided evidence of a protective action of the
above-mentioned molecules against O3
stress (Loreto and
Velikova 2001). Cyanide, a secondary product associated
with ethylene formation, is considered in sensitive plants to
be an ulterior cause for necrosis (Grossmann 1996).
Acute O3
exposure negatively affects the photosynthetic
performance of the plant, mainly by inhibiting the
functionality of the photosystems (PS). Strong reduction in
photosynthetic activity, accompanied by a drop in stomatal
conductance, were observed in both ozone-sensitive and
ozone-tolerant tobacco cultivars after one single acute
fumigation (300 ppb during 3 h); these reductions were
reversible in the tolerant cv. and irreversible in the sensitive
one, demonstrating how damage caused to the PS can be
relatively easy to recover in tolerant plants (Pasqualini
et al 2002a). The effects of O3
and fungal pathogens
on photosynthesis are similar and, in both cases, affect
mainly PS-II, but some differences exist. Inhibition of
photosynthesis induced by O3
and by a necrotrophic fungal
pathogen, Pleiochaeta setosa, was compared in white lupin
leaves by chlorophyll imaging. In both cases, PS-II was the
main target of the perturbations, but the damage caused by
O3
became evident in a significantly shorter time than in
the case of the fungus; moreover, the spatial patterns of the
response on the surface of the leaves were totally different
for the two elicitors (Guidi et al 2007).
3.2 Chronic damage
This type of damage is caused by long-term exposure to low
O3
concentrations; a reduction in fitness and competitiveness
Paolo Zuccarini4
J. Biosci. 34(1), March 2009

5. are usually associated with it. The most common symptoms
are premature senescence, alteration in the metabolism of
sugars, inhibition of photosynthesis, loss of balance in the
redox status and production of ROS in the stroma (Pell et
al 1997).
Acceleration of leaf ageing is the most typical response
of plants to exposure to chronic doses of O3
(Pell and Dann
1991; Ljubešić and Britvec 2006), and is associated with
an increased production of ethylene (Tingey et al 1976). No
cell death occurs, since the concentration of the oxidizing
agents is low enough to be tolerated by the plasmatic
membrane; nevertheless, free radicals tend to accumulate
over time, since the detoxifying enzymes of the plant
cannot completely “clean” them. This shows that ageing is
connected to an increase in oxidizing events and a reduction
in the capacity to counter them (Sohal and Weindruck
1996). There is a link between leaf ageing and trends in
the concentration of Rubisco in plant tissues. As a matter
of fact, treatment with O3
causes a reduction in the peaks
of Rubisco (Dann and Pell 1989; Pell et al 1992, 1994;
Kopper and Lindroth 2003) and accelerates the degradation
of the protein (Eckardt and Pell 1994); this suggests that
the drop in Rubisco levels, and its consequent decline in
photosynthesis, can be one of the ways through which O3
causes foliar senescence.
Ethylene is often associated with senescence too, but
its precise role is not yet clear, apart from the fact that
it is a promoter (Miller et al 1999). The most accepted
hypothesis considers ethylene to have a direct role both
in accelerating the process of senescence (Reid 1989) and
in acting as a signalling molecule (Guo and Ecker 2004;
Setyadjit et al 2004). The role of ethylene in the process of
senescence is closely related to that of polyamines (Pandey
et al 2000). The actions of these two classes of molecules
are sometimes complementary and sometimes antagonistic,
depending on the specific physiological phase undergone by
the plant. Yang et al (2008) provided evidence that ethylene
plays a key role in the regulation of several developmental
processes connected to leaf senescence, such as petal
necrosis and corolla abscission on transgenic Nicotiana
sylvestris specimens. Woltering et al (2002) showed on
tomato plant cells how the action of ethylene in inducing
programmed cell death during leaf senescence can be
enhanced by the concomitant application of camptothecin,
an inducer of apoptosis, but the supply of ethylene alone did
not lead to significant alterations. Karaivazoglou et al (2004)
monitored the production rates of ethylene during ripening
and senescence of tobacco leaves, showing how an increase
in ethylene production coincided with the first symptoms of
leaf senescence such as chlorophyll breakdown and decrease
of dry weight; the concentration peak was reached about one
week after the start of the process and was 5–6-fold higher
than basal ethylene production.
Alteration of sugar metabolism occurs in very sensitive
plants (such as deciduous trees), with accumulation of
starch in the guard cells and decrease of starch in the
mesophyll (Günthardt-Goerg et al 1997). The accumulation
of starch and other hexoses inhibits the activity of several
enzymes involved in the Calvin cycle and photosynthetic
efficiency is reduced (Krapp et al 1993). The reduced CO2
fixation increases the pool of reducing equivalents, with the
consequent direct reduction of O2
by PS-I (Mehler reaction).
Therefore, the loss of balance in the redox status is the cause
of liberation of ROS into the stroma (Melhorn et al 1990;
Mittler et al 2004).
Chronic O3
exposure is commonly known to affect plant
photosynthesis (Heath 1994), and this involves a variety
of mechanisms that can act separately or in combination,
depending on the plant species and on the conditions of
exposure. A central role in the inhibition process is played
by the stomata (Inclán et al 1998), the partial closure of
which represents the most immediate form of response, but
subsequent metabolic regulations follow, bringing about
a general limitation of physiological performance and a
decline in plant productivity.
Chloroplasts are the most important targets of chronic
O3
exposure: characteristic symptoms are represented
by alterations in their size and functionality, and in the
composition of the stroma. Both O3
fumigations and
natural tropospheric concentrations induced significant
size reductions in the chloroplasts of needles of Scots pine
and Norway spruce, and an increase in the electron density
of the stroma, especially on the upper side of the leaves
(Kivimäenpää et al 2005). Serious chloroplast injuries due to
oxidative stress were observed in sensitive clones of Betulla
papyrifera after O3
exposure, with subsequent dramatic loss
of functionality; no ultrastructural injuries were observed in
tolerant clones, as O3
-elicited H2
O2
production is restricted
to the apoplast (Oksanen et al 2004).
A typical response of plants to chronic exposure to O3
is
represented by a decrease in the quantum yield of electron
transport. This strategy allows the plant to reduce the
photosynthetic assimilation rate at analogous conditions of
irradiation, and is probably intended to reduce adenosine
triphosphate (ATP) and nicotinamide adenine dinucleotide
phosphate (NADPH) production in order to put the plant in
equilibrium with the decreased demand for the Calvin cycle
subsequent to O3
stress. Evidence of this fact is provided by
a trial in which chronic O3
fumigation on poplar (60 nl l−1
for
5 h day−1
over 15 days) resulted in a significant reduction in
the CO2
assimilation rate, due not only to strong stomatal
closure but also to limitation of the dark reactions of the
photosynthetic process, and the connected downregulation
of photosynthetic electron transport (Guidi et al 2001).
As can be seen, a problem for plants exposed to chronic
O3
concentrations is to reduce the photosynthetic rate, tune
Tropospheric ozone as a fungal elicitor 5
J. Biosci. 34(1), March 2009

6. Paolo Zuccarini6
J. Biosci. 34(1), March 2009
it with the changed metabolic demands, and put into action
different alternative strategies for reducing equivalents.
Another possible mechanism of the photosynthetic
response can in fact involve more precocious steps, such
as the inhibition of PS-II, with no alterations in quenching
parameters (Guidi et al 2001). Ranieri et al (2001) showed
in poplar how chronic fumigation with O3
can induce
alterations in tylakoid functionality and composition; the
activity of both the photosystems (PS-II and PS-I) was
significantly reduced, and so was the concentration of all
the polypeptides analysed. This provides evidence of the
fact that, at a chronic level, O3
generally inhibits the activity
of the electron transport chain by lowering the PS protein
and pigment content, and all of these are strategies to
reduce the rate of photosynthetic activity to face the adverse
conditions.
Some similarities exist between the effects caused on
the photosynthetic process by chronic O3
exposure and
fungal pathogens. Carter and Knapp (2001) analysed a
large amount of published and unpublished data to show
that, among others stressors, fungal pathogens and O3
cause
alterations in the optical properties of leaves at almost the
same wavelengths.
The results shown provide evidence of an interaction
between the primary metabolism and the possible responses
to O3
stress, causing changes in the cell biochemistry,
structure of chloroplasts and composition of their proteins,
levels of reducing substances (such as nicotinamide adenine
dinucleotide [NADH] and other reduced equivalents) and
the redox balance of glutathione and ascorbate in the stroma.
These changes can affect plant productivity, shift certain
metabolic pathways (e.g. the shikimate way, Schmid and
Amrhein 1995) and alter the capacity of the plant to react to
future biotic and abiotic stresses.
O3
seems complexively to not cause direct damage
to the plant, as already theorized by Heath (1994), but to
activate several signal pathways in it. In this sense, O3
takes
shape of an abiotic elicitor, capable of stimulating plant
reactions that are similar to the ones derived from pathogen
attacks. These responses can either cause damage or act as
the basis for SAR; the discriminating factors are mainly the
intensity of the exposure and the individual sensitivity of the
subject.
4. Ozone as a fungal elicitor
In the previous section, plant responses to O3
stress
were analysed from the point of view of the kind of
damage induced. This section highlights all the cases
in which O3
elicits reactions comparable to the ones
generated by a fungal pathogen, potentially inducing the
plant to resist abiotic stresses. This mechanism is called
cross-induction.
4.1 Phytoalexins
Several experiments conducted during the past 30 years
demonstrate the capacity of O3
to stimulate the production
and accumulation of phytoalexins in different species.
O3
induces accumulation of isoflavonoid phytoalexins
in soybean plants (Keen and Taylor 1975); of stilbenic
phytoalexins in pine (Sandermann 1996; Chiron et al 2000)
and grape (Schubert et al 1997) (the induction occurs at the
level of transcription, Zinser et al 1998); of catechins in
spruce and pine (Koricheva et al 1998); of the phytoalexins
of furanocoumarin in basil (De Moraes et al 2004). In
conifers, catechins and stilbenes can be accumulated and
stored for several months (Langebartels et al 1998), a
phenomenon called “memory of the ozone stress”.
The molecular mechanisms through which O3
induces
phytoalexyn biosynthesis and the genes involved are still
an object of investigation. In leaves of Phaseolus vulgaris
L. exposed to realistic O3
doses, an increase was observed
in RNA accumulation of phenyalanine ammonia lyase,
naringenin chalcone synthase and chalcone isomerase
genes. The substances produced by these genes are involved
in the synthesis, among others, of isoflavonoid phytoalexins
(Paolacci et al 2001). Grimmig et al (2004) provided
evidence of the fact that at least two different signalling
pathways for O3
-induced gene expression are involved, one
depending on ethylene and the other an independent one.
The role of O3
in inducing phytoalexyn biosynthesis
can also be exploited in the control of postharvest decay
of fresh fruit. In 1997, an expert panel declared O3
to be
generally considered as safe (GRAS) for applications
involving food contact (US FDA 1997). In table grapes, O3
was demonstrated to induce resveratrol and pterostilbene
phytoalexins, providing better resistance of the berries to
subsequent infections with Rhizopus stolonifer (Sarig et al
1996).
4.2 Cellular barriers
O3
exposure can provide plants with a reinforced cellular
structure, particularly by strengthening the cell walls. This
effect is generally observed at foliar level and is connected
in most cases with an enhancement of lignin production and
deposition, and with the switching of the related metabolic
pathways towards the biosynthesis of particularly resistant
and flexible species of lignin.
O3
induces, usually at transcriptional level, the activity
of cinnamyl alcohol dehydrogenase (Zinser et al 1998;
Soldatini et al 2005) leading to the production of a lignin
with juvenile characteristics that is closely correlated
to the extensins (Lange et al 1995). This modification
results in more resistant cells, with more lignified and
elastic walls. In parsley (Eckey-Kaltenbach et al 1994)

7. Tropospheric ozone as a fungal elicitor 7
J. Biosci. 34(1), March 2009
and tobacco (Sandermann 1996), O3
induces the synthesis
of callose. In soybean plants, O3
treatment induces
modifications in the metabolism of phenylpropane and in
the phenolic composition of leaves, leading to a higher
content of hydrocinnamic acid, lignin and suberin. This
was, again, mainly due to the increase in cinnamyl alcohol
dehydrogenase activity, but O3
also elicited reactions
typically associated with wound responses and browning
(Booker and Miller 1998).
O3
fumigation of poplar trees induced significant
increase in the foliar activities of shikimate dehydrogenase,
phenylalanine ammonia lyase and cinnamyl alcohol
dehydrogenase, enzymes involved in various steps of the
metabolic pathway for the biosynthesis of lignins. A higher
proportion of Klason lignin was observed in extract-free
leaves of treated plants, and the lignins synthesized in
response to O3
showed a different structure with regard to
pre-existing lignins, with more juvenile characteristics such
as enrichment of carbon–carbon interunit bonds and in p-
hydroxyphenylpropane units (Cabane et al 2004).
The data shown here suggest that O3
fumigation can
provide the plant with higher tolerance to O3
itself and
to fungal pathogens by inducing, among other effects, a
substantial reinforcement of the cell wall by synthesis of
higher amounts of lignins that can provide better mechanical
performance than constitutive ones.
4.3 PR proteins
Fungal and viral infections are responsible for an increase
in PR proteins among the soluble protein fraction of leaves
of most plants (Bol et al 1990; Bowles 1990); O3
is capable
of triggering the same response. The common factor in
the stimulation, in the case of both O3
stress and attack by
pathogens, is the liberation of ethylene (Sandermann 1996;
Van Loon et al 2006), and the molecules whose production
is stimulated more are glucanase, chitinase and glutathione-
S-transferase-1.
Both the stimulation and increase of pre-existing
production of PR proteins have been demonstrated in
tobacco (Ernst et al 1992; Ernst et al 1996; Yalpani et al
1994; Van Buuren et al 2002), parsley (Eckey-Kaltenbach
et al 1997), Arabidopsis (Sharma and Davis 1994; Conklin
and Last 1995; Sharma et al 1996; Lim et al 2003) and
spruce (Kärenlampi et al 1994) treated with O3
. RNA-blot
analysis performed on O3
-tolerant and O3
-sensitive clones
of hybrid poplar showed that O3
-induced mRNA levels of
O-methyltransferase, a PR protein, were significantly higher
in the O3
-tolerant clones (Riehl Koch et al 1998). Long-term
induction of genes encoding stress-related proteins PR-10
and PAL was related, in birch, to macroscopic symptoms of
injury (necrotic flecks) and enhanced yellowing of leaves,
and leaf injuries were connected with short-term stomatal
closure response in a highly complex manner (Pääkkönen
et al 1998). O3
-induced production of PR proteins can be
enhanced by the concomitant action of other abiotic factors:
in potato plants susceptible to Phytophthora infestans, an
increase in the constitutive activities of the PR proteins
β-1,3-glucanase and osmotin is mediated by the combined
action of high O3
and CO2
concentrations, resulting in an
improved resistance to the pathogen (Plessl et al 2007).
4.4 Signal substances
These substances have a role in transmitting information
from the areas attacked by O3
to the rest of the plant to
activate the defences of the organism, such as PR proteins.
The message can be carried by a hypothetical O3
receptor, or
redox-sensor; by the oxidative burst at apoplastic and, later,
at symplastic level (Ernst et al 1992; Sharma and Davis
1994) or by other messengers.
The two most important signal molecules are ethylene and
SA; they can either be induced directly by O3
or be secondary
messengers. It was demonstrated that these two molecules
can act in concert to influence cell death in O3
-sensitive
genotypes and that, at the same time, O3
-induced ethylene
production is dependent on SA (Rao et al 2002), and SA
production is regulated by ethylene (Ogawa et al 2005)
The production and circulation of ethylene as a
consequence of O3
stress has been studied in potato (Pell et
al 1997; Schlagnhaufer et al 1998; Sinn et al 2004), tomato
(Tuomainen et al 1998; Moeder et al 2002), Arabidopsis
(Overmeyer et al 2000) and birch (Vahala et al 2003),
and is testified by the activation of 1-aminocyclopropyl-
1-carboxylic acid (ACC)-synthase and ACC-oxidase (Yin
et al 1994; Glick et al 1995; Sandermann 1996; Moeder
et al 2002). The induction of ACC-oxidase transcription is
the fastest response to O3
in plants, occurring in less than
30 min from stimulation (Pell et al 1997; Tuomainen et al
1997). In tomato, exposure to O3
concentrations of 85 nl l-1
for 5 h caused visible foliar damage by 24 h, and the activity
of ACC-synthase started to increase after 2 h (Tuomainen et
al 1997); however, in tomato, changes in mRNA levels of
specific ACC-synthase, ACC-oxidase,and ethylene receptor
genes occurred within 1–5 h of treatment (Moeder et al
2002). The effects produced by stress-induced ethylene are
typical of both HR and SAR reactions, such as synthesis of
PR proteins (Ernst et al 1992; Ernst et al 1996), synthesis
of stilbene synthase (Schubert et al 1997), accelerated
senescence (Pell et al 1997), inactivation of Rubisco (Glick
et al 1995) and modulation of programmed cell death (Lamb
and Dixon 1997; Greenberg 1997). Some studies also
suggest that ethylene may react non-enzymatically with O3
to give a superoxide radical, thereby directly determining
the responses of plants to O3
(Elstner et al 1985; Mehlhorn
and Wellburn 1987).

8. Paolo Zuccarini8
J. Biosci. 34(1), March 2009
SA is considered a signal molecule capable of inducing
both HR and SAR responses (Lamb and Dixon 1997;
Durner et al 1997; Takashi et al 2006). Studies demonstrate
the induction of SA and of its β-D-glycosidic conjugate
in tobacco (Yalpani et al 1994) jointly with an increase
in resistance to Tobamovirus, and on Arabidopsis,
with a concomitant induction of resistance to Pseu-
domonas syringae (Sharma et al 1996). SA also
has a role of messenger similar to that of ethylene,
mediating SAR responses such as induction of PR proteins
and lipoxygenases involved in the synthesis of jasmonic
acid, which prevents the visible symptoms of O3
stress
(Ernst et al 1992; Thalmair et al 1996; Eckey-Kaltenbach et
al 1997; Sharma et al 1996). In some genetically modified
organisms containing a bacterial salicylate hydroxylase,
SAR responses are significantly affected (Sharma et al
1996; Örvar et al 1997). It is commonly accepted at present
that high SA content could trigger the production of ROS
with subsequent SA-mediated cell death (Pasqualini et al
2002b).
4.5 Antioxidative systems
An important effect of O3
as an elicitor is to stimulate
the synthesis and accumulation of several antioxidative
enzymes located in the apoplast and plasmatic membrane,
such as catalases, glutathione peroxidases, glutathione-
S-transferases (Sandermann 1996; Noormets et al 2000),
superoxide dismutase and ascorbate peroxidase; the latter
two usually have light and delayed effects (Willekens
et al 1994). The kind and severity of the antioxidative
response depends on the plant species, on the onthogenic
phase (Sandermann 1996; Heath and Taylor 1997) and on
the compartments involved (cytosol, chloroplast, apoplast)
(Sandermann 1996; Schraudner et al 1997; Van Hove et
al 2001), since each of them hosts different antioxidative
systems.
Ethylene has been shown to have an important role in
inducing HR in hypersensitive tobacco (Greenberg 1997);
when sensitive Arabidopsis was deprived of ascorbic acid
the noxious effect of ROS was detected (Conklin et al 1996).
Ascorbate has been studied for its detoxifying properties in
spinach (Luwe et al 1993), which plays a role both as a direct
antioxidant and reducer of α-tocopherol that is activated in
this way, and in soybean (Robinson and Britz 2001), in
which it was shown to play a more important role than
dehydroascorbate in enhancing plant tolerance to elevated
levels of O3
. Polyamines, both in their free and conjugated
forms, have been demonstrated to reduce the gravity of
lesions due to O3
in tobacco (Kangasjärvi et al 1994) by
inhibiting lipid peroxidation and preventing premature
senescence, and regulating adaptation of the photosynthetic
apparatus.
4.6 Other abiotic elicitors
Tropospheric O3
has been demonstrated to play an important
role as a fungal elicitor, but it is not the only chemical with this
action. Evidence has been collected over many years of the
possibility that other abiotic factors could trigger mechanisms
of plant reaction similar to the ones provoked by bacterial or
fungal pathogens. This is due to the fact that O3
and other
abiotic stressors can, in specific cases, trigger analogous
metabolic mechanisms of response in the attacked plant, most
of which are mediated by the production of ROS, providing the
opportunity for interesting crossed applications. For example,
wounding prior to high exposure to O3
of tobacco reduced the
severity of injury caused by O3
, because of overexpression
of the antioxidant enzyme ascorbate peroxidise due to the
mechanical stress (Örvar et al 1997).
With regard to agents other than O3
, a variety of biotic
and abiotic elicitors for the production of phytoalexins have
been identified (Darvill and Albersheim 1984). Davis et al
(1986) demonstrated on cotyledons of soybean plants that
the accumulation of phytoalexins, a typical plant response
to microbic aggressions, is favoured by the combined and
synergistic action of the elicitor-active hexa-β-glucosyl
glucitol, and various biotic and abiotic elicitors. Treatment of
cotyledons of Vicia faba with both ultraviolet (UV) radiation
and freezing–thawing caused a remarkable increase in the
production of phytoalexins, particularly wyerone, giving
results comparable with those caused by a typical biotic
agent such as Botrytis cynerea (Soylu et al 2002).
Low-energy ultrasound (US) was demonstrated to
induce plant defence responses and increase the production
of several secondary metabolites in Panax ginseng cells
in suspension culture, effecting an elicitor-like effect.
In particular, increased cross-membrane ion fluxes and
production of ROS were observed, as well as synthesis of
saponins (Wu and Lin 2002).
There is also evidence of overlap of the effects of O3
and other stressors, both biotic and abiotic, on plants. O3
treatment on parsley cell cultures resulted in simultaneous
induction of the pathways of phenylpropanoid metabolism,
usually associated with the action of fungal elicitors and
UV irradiation, respectively (Eckey-Kaltenbach et al 1994),
demonstrating how this gas can elicit a wide range of defence
responses in plants. Yalpani et al (1994) showed how both
O3
and UV light stimulated the production and accumulation
of SA and PR proteins in tobacco, increasing the resistance
against tobacco mosaic virus.
O3
is therefore not the only agent capable of inducing
plant defence responses similar to those due to fungal attack.
It shares this activity with numerous biotic and abiotic
factors, but stands out for its efficacy (Sandermann 1996,
2004) and the wide applicability of this property. This is
the reason why O3
fumigation is used successfully today

9. Tropospheric ozone as a fungal elicitor 9
J. Biosci. 34(1), March 2009
in a variety of agronomical applications, such as conferring
resistance against fungal pathogens; for example, against
Bipolaris sorokiniana in barley and fescue, against Phoma
lingam in rape (Płazek et al 2001) or against Botrytis cinerea
in strawberry plants (Nadas et al 2006).
5. Conclusions
The data presented here demonstrate how O3
shows the
typical characteristics of a fungal elicitor, which can be
utilized both with the objective of inducing resistance to
the attack of pathogens in plants and for the study of plant
defence reactions to the above-mentioned attacks. This
idea is feasible by virtue of the fact that O3
application is
economically convenient and technically easy to perform.
O3
can be used alone or in association with other
preparations such as +active pathogens, fungal elicitors and
signal substances. In particular, O3
and ethylene are the only
elicitors that can be easily removed after each experiment.
O3
is the best among various substances for performing
the treatment, since it is the easiest to produce, apply and
remove. Several examples exist in the literature of the use of
O3
to induce resistance to fungal pathogens.
In conclusion, O3
is an important instrument for the study
of plant responses to biotic and abiotic stress, and a valid
alternative to more expensive and complicated treatments
for the induction of resistance to several pathogens, with no
particular environmental impact. However, this subject has
not been studied deeply enough yet, since each plant can
show a different set of responses to different applications
of O3
; moreover, the constant increase in tropospheric O3
in several parts of the world is causing a huge change on a
global scale. The two-way activity of O3
, which is capable of
both predisposing plants to the attack of viruses, pathogens
and insects, and inducing resistance to these same factors,
depending on factors such as the kind of plant and the nature
of the exposure, makes us realize how the medium- and long-
term effects of this phenomenon are not easily predictable.
Further, capillary experimentation will be required.
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MS received 27 November 2007; accepted 3 November 2008
ePublication: 6 January 2009
Corresponding editor: VIDYANAND NANJUNDIAH