Signalling and cell death in ozone-exposed plants



    Corresponding author
    1. Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, POB 56 (Viikinkaari 9), FI-00014 Helsinki, Finland
      Jaakko Kangasjärvi. Fax: + 358 9191 59552; e-mail:
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    1. Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, POB 56 (Viikinkaari 9), FI-00014 Helsinki, Finland
    Search for more papers by this author

    1. Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, POB 56 (Viikinkaari 9), FI-00014 Helsinki, Finland
    Search for more papers by this author

Jaakko Kangasjärvi. Fax: + 358 9191 59552; e-mail:


Experiments with Arabidopsis mutants and sensitive and tolerant pairs in several other species have elucidated the molecular basis of plant ozone sensitivity and ozone lesion development. They have indicated an important role for hormonal signalling in determining the outcome of ozone challenge at the cellular level. The reactive oxygen species (ROS) from ozone degradation can cause either direct necrotic damage or induce the process of programmed cell death. Perception of ozone or ROS from its degradation in the apoplast activates several signal transduction pathways that regulate the responses of the cells to the increased oxidative load. Plant hormones salicylic acid, jasmonic acid, ethylene and abscisic acid are involved in determining the duration and extent of ozone-induced cell death and its propagation. Salicylic acid is required for the programmed cell death, ethylene promotes endogenous ROS formation and lesion propagation, and jasmonic acid is involved in limiting the lesion spreading. Abscisic acid is most likely involved through the regulation of stomata and thus is expected to affect lesion initiation. The roles and interactions of perception of ozone, the immediate downstream responses, hormone biosynthesis and signalling during ozone lesion initiation and formation are reviewed.


abscisic acid




1-aminocyclopropane-1-carboxylic acid


ACC synthase




Arabidopsis thaliana respiratory burst oxidase homologue


Arabidopsis thaliana ecotype Columbia

CTR/ctr, constitutive

triple response PROTEIN/mutant


drought-responsive element binding protein


ethylene insensitive PROTEIN/mutant


ethylene overproducer PROTEIN/mutant


ethylene-resistant (ethylene receptor) PROTEIN/mutant


G-protein α subunit gene


hypersensitive response


jasmonic acid


jasmonate resistant PROTEIN/mutant


jasmonate insensitive PROTEIN/mutant


methyl jasmonate


mitogen activated protein kinase




Pseudomonas putida salicylate hydroxylase gene




programmed cell death


radical-induced cell death PROTEIN/mutant


reactive oxygen species


salicylic acid


salicylic acid-induced protein kinase


wounding-induced protein kinase.


Ozone (O3) is the triatomic form of oxygen. It is an important protective component against UV radiation in the stratosphere, but in the troposphere it is one of the most notorious air pollutants. In sensitive plant species and cultivars, long-term ozone exposure of relatively low concentrations causes a reduction in photosynthesis and growth, as well as premature senescence. These in turn cause decreased growth and crop yields, as well as decreased pathogen tolerance and other possible ecological alterations (Heath & Taylor 1997; Pell, Schlagnhaufer & Arteca 1997; Langebartels & Kangasjärvi 2004). In contrast to the fairly subtle effects of chronic ozone exposure, a short, high-level exposure causes visible O3-lesions on leaves, the appearance of which resembles hypersensitive cell death. Research during recent years has shown that the similarity of these processes is not only external, as reviewed in Kangasjärvi et al. (1994) and Rao, Koch & Davis (2000a); these phenomena share many physiological and molecular features.

In our previous review (Kangasjärvi et al. 1994) the molecular responses of plants to ozone were mostly treated by comparing the known physiological responses of plants to O3 with the then-known information arising from plant–pathogen interactions – 10 years ago there were only a few articles reporting molecular responses of plants to ozone and the basic mechanisms of O3-induced cell death. Now we review recent research on the mechanisms involved in plant sensitivity to acute, high-level O3. A huge amount of information directly relevant to the topic has become available during the past 10 years and it is not even possible to include all the recent developments on various aspects of ozone biology in plants in one review. Therefore we will restrict this review only to the mechanisms of plant ozone sensitivity defined as lesion formation and dissect it to the following six components: (1) regulation of O3 flux to leaves; (2) O3 degradation to reactive oxygen species (ROS) and detoxification by ascorbate (ASC) in the apoplast; (3) O3 sensing/perception; (4) O3-induced active production of ROS; (5) early activation of mitogen-activated protein kinases by O3; and (6) hormonal regulation of O3-induced lesion formation. We discuss the processes involved and concentrate especially on experiments addressing and identifying molecular mechanisms responsible for O3 responses at the cellular level. Most of the research reviewed here has been performed using model plants and particularly mutants of Arabidopsis thaliana. This has expanded our knowledge about the basis of plant O3 sensitivity. Several recent reviews that discuss plant responses to ozone from many different perspectives not covered here are available (Heath & Taylor 1997; Langebartels et al. 1997, 2002a, b; Sandermann et al. 1998; Kley et al. 1999; Rao & Davis 2001; Mahalingam & Fedoroff 2003; Conklin & Barth 2004; Langebartels & Kangasjärvi 2004; Timonen, Huttunen & Manninen 2004; Baier et al. 2005; Foyer & Noctor 2005).


Since ozone entry through the leaf cuticle is negligible (Kerstiens & Lendzian 1989), stomata play a fundamental role in determining the flux of ozone into the apoplastic space. Their sensitivity to external stimuli is an important factor in the overall plant sensitivity/tolerance to ozone. If the stomatal aperture is wider, and consequently the flux of gaseous substances is higher in one plant when compared with another, the dose of ozone delivered to the intercellular space is also larger (Heath & Taylor 1997; Sandermann & Matyssek 2004).

Stomatal closure in response to ozone is regarded as a protective mechanism and has been documented several decades ago (Hill & Littlefield 1969). However, it is still mostly unresolved how O3 affects the stomatal function. The mechanisms suggested span from changes in photosynthesis to abscisic acid (ABA) signalling, generation of an ‘artificial’ ROS burst directly in the guard cells, and ethylene emission, which all affect stomatal aperture. Moldau, Sober & Sober (1990) showed that O3 induced rapid stomatal closure already 12 min after the onset of the exposure. At the same time there was no change in the efficiency of carboxylation measured as mesophyll conductance to CO2. On the basis of this Moldau et al. (1990) concluded that O3-induced stomatal closure was rather a direct effect of O3 on guard cells than a result of decreased mesophyll photosynthesis. On the other hand, Martin et al. (2000) modelled the data from earlier literature and showed that much of the stomatal response caused by acute O3 can be predicted by changes occurring in the mesophyll photosynthesis. Involvement of photosynthesis in O3-induced stomatal responses can also be predicted from the recent experiments by Joo et al. (2005) who showed that oxidative burst generated by O3 was first detectable in the chloroplasts of guard cells.

O3-induced ethylene emission has also been shown to cause stomatal closure (Gunderson & Taylor 1991). Involvement of ABA signalling in O3-induced stomatal closure is demonstrated by a comparison of the wild-type Arabidopsis Col-0 to ABA insensitive mutants; O3-induced stomatal closure was significantly faster in the wild type than in the mutants (Ahlfors et al. 2004a). Eventually, however, O3 caused the closure of stomata in ABA insensitive mutants indicating that O3 affects stomata also by ABA-independent mechanisms.

It has been shown that ABA-dependent closure of stomata is ROS-mediated (Pei et al. 2000; Murata et al. 2001; Zhang et al. 2001). Therefore also O3– or the ROS formed from the degradation of O3 in the apoplast – could have direct effect on stomatal function. The activity of the Arabidopsis NADPH oxidases ATRBOH D and ATRBOH F (Arabidopsis thaliana Respiratory Burst Oxidase Homolog), which generate ROS during hypersensitive response, is required for elevation in [Ca2+]cyt in guard cells, which in turn triggers the closure of the stomata. The ROS formed from O3 degradation could affect stomata either by acting as an ‘artificial’ replacement for the function of the ABA-induced ROS, or by affecting (unidentified) components in the perimeter of the guard cells and eliciting downstream signalling responsible for the activation of the NADPH oxidase. ROS also affect two protein phosphatases, ABI1 and ABI2, which are negative regulators of ABA signalling; H2O2 inhibits the activity of these proteins (Meinhard & Grill 2001; Meinhard, Rodriguez & Grill 2002). Thus, this is one way in which ROS, and possibly also O3 affect (the ABA-dependent) plasma membrane Ca2+ influx. It has also been shown that O3 can affect the stomatal opening directly by regulating the K+ fluxes in the guard cells (Torsethaugen, Pell & Assmann 1999). However, only a few reports have addressed the effect of O3 on ion fluxes across the plasma membrane in general (Castillo & Heath 1990; McAinsh et al. 1996; Clayton et al. 1999) and only one in the stomatal guard cells specifically (Torsethaugen et al. 1999), thus the mechanisms by which O3 affects the stomatal function require further study.


The O3 concentration inside the leaf during ozone exposure is close to zero (Laisk, Kull & Moldau 1989), which means that it is rapidly degraded in the apoplast, and thus the apoplastic antioxidative capacity is the next factor in determining the fate of the leaf cells. Ascorbate provides important protection from oxidative injury by removing the harmful ROS generated from ozone (Luwe, Takahama & Heber 1993). The protective role of ASC is also supported by the enhanced O3 sensitivity of mutants deficient in ascorbate biosynthesis (Conklin, Williams & Last 1996) and by the observation that transgenic ascorbate peroxidase anti sense tobacco plants are more sensitive to O3 (Örvar & Ellis 1997).

The role of the apoplastic ascorbate as a protective mechanism against O3 is not, however, simple and clear-cut. For example, Sanmartin et al. (2003) showed that removal of the reduced ASC from the apoplast by overexpressing apoplastic ascorbate oxidase made plants substantially more sensitive to O3. On the other hand, it has been shown that, at least in wheat and barley, the removal of O3 by direct reaction with apoplastic ASC is not of major significance since mesophyll cell walls are thin and the effective path length for the reaction is short (Kollist et al. 2000). Furthermore, in an attempt to examine the in vivo reactivity of ASC with O3, ASC was not able to inhibit the O3-mediated oxidation of fluorescent dyes pre-infiltrated into the apoplast (Jakob & Heber 1998). However, since apoplastic ASC is also a natural substrate for peroxidases and oxidases present in this compartment of the cell it is possible that ASC pre-infiltrated into the apoplast was oxidized before the O3-treatment. Furthermore, since ASC is synthesized inside the cell, in addition to its use by apoplastic peroxidases and oxidases, its transport rate across the plasma membrane must be taken into account when discussing the antioxidative capacity of apoplastic ASC in the detoxification of O3. Padu et al. (2005) analysed ASC use by apoplastic peroxidases and oxidases in silver birch (Betula pendula) trees growing under the ambient and double ambient concentrations of CO2 and O3 and showed that capacity of the plasma membrane/cytosol system to supply ASC was not exceeded in any of the treatments, even when the stomata were fully open and the O3 flux to the mesophyll cell walls was at its maximum. Furthermore, comparison of O3 flux into the leaf with ascorbate flux through the plasma membrane indicated that ascorbate flux through the plasma membrane was more than three times higher than flux of O3 reaching the plasma membrane (Kollist et al. unpublished) Thus in silver birch, at least under low, current ambient concentrations of O3, the apoplastic ascorbate concentration as such does not seem to be the limiting factor in the detoxification of O3, or the ROS from O3 degradation in the apoplast. A recent review (Conklin & Barth 2004) gives a contemporary description on the role of ascorbate in plant O3 responses.


It has been suggested that O3 is perceived in the apoplast (Kangasjärvi et al. 1994). Yet, how cells sense or perceive the apoplastic reactive oxygen species is still mostly unknown. Since O3 elicits responses in the cells and causes drastic changes in the global gene expression, even when no visible damage is evident (Mahalingam et al. 2003, 2005; Tamaoki et al. 2003b, 2004; Tuominen et al. 2004), ozone, or its breakdown products must be sensed by the cells to induce the changes observed in gene expression. Three different mechanisms could be involved in O3/ROS sensing: (a) perception by an apoplastic receptor protein, which is directly modified by the ROS generated from O3 breakdown, or which senses O3/ROS-induced modifications in another component present in the apoplast (see Baier et al. 2005); (b) oxidation of plasma membrane lipids, which results in the formation of lipid-based signalling molecules that are further sensed; or (c) a change in the total cellular redox balance as a result of action of O3/ROS on redox-active substances, such as ascorbic acid, glutathione or the total cellular NAD(P)H/NAD(P). These alternatives cannot be regarded as exclusive since they may all be involved in the various individual responses elicited by O3 in the cells affected.

Membrane-associated receptors and G-proteins

It has been shown that membrane receptor-mediated signalling could be involved in oxidant signalling in tobacco suspension-cultured cells (Miles, Samuel & Ellis 2002). The use of a membrane non-permeable reagent that interferes with membrane receptor-mediated signalling prevented the activation of tobacco Salicylic Acid-Induced Protein Kinase (SIPK) by ultraviolet C radiation, O3 and H2O2. It was also shown that the UV-C-induced activation of SIPK was ROS-dependent since the free radical scavengers used in the experiments completely abolished the MAPK activation by UV-C radiation. Furthermore, activation of AtMPK6, the Arabidopsis homologue of SIPK, required both ROS and Ca2+-influx, but did not involve either the α or β subunit of the heterotrimeric G-protein (Miles et al. 2004), a membrane-bound GTPase that transduces extracellular cues to intracellular signals in several different processes in eukaryotic organisms. Similarly the up-regulation of Arabidopsis AtMPK3 gene was independent of G-protein (Joo et al. 2005). In plants, G-proteins are thought to be involved in interactions of plant hormones and plant defence responses to wounding and pathogen infection (Assmann 2002). Booker et al. (2004) showed that insertion mutants of Arabidopsis canonical G-protein α subunit gene (GPA1) were more tolerant to low-level O3 than the wild-type Col-0 in long-term O3-exposures. This suggests that these null mutants had altered perception of O3 or downstream signals. Similarly, it was shown that in short-term acute O3 exposures Arabidopsis plants with null mutations in the genes encoding the α and β subunits of the single heterotrimeric G protein were less and more sensitive, respectively, to O3 damage than wild-type Col-O plants (Joo et al. 2005).

G-proteins are involved in guard cell ABA signalling (Wang et al. 2001; Coursol et al. 2003; Pandey & Assmann 2004). As discussed above, stomatal conductivity might be one of the factors affecting plant sensitivity to ozone. Knocking out the putative G-protein coupled receptor GCR1, rendering plants hypersensitive to ABA in respect to stomatal responses, has been shown to interact with GPA1 (Wang et al. 2001). These plants were also more drought tolerant than the wild type, probably due to their lower transpiration rates. However, this was not reflected in the ozone sensitivity of the GCR1 null mutants, which did not differ from the wild type (Booker et al. 2004). This suggests that the role of G-proteins in O3-related processes is separate from their role in stomatal regulation.

It has been shown in rice that the activation of the plasma membrane NADPH oxidase during the hypersensitive response (HR) requires the small GTPase Rac since constitutively active Rac-induced active ROS production and ROS-dependent cell death in both cultured rice cells and leaves of transgenic plants (Kawasaki et al. 1999). Rac, in turn, is activated through the G-protein α subunit (Suharsono et al. 2002). Similarly, the O3-induced activation of NADPH oxidase and the subsequent oxidative burst (which is reviewed below) is mediated through the action of G-protein (Joo et al. 2005). Thus, the interactions between ABA signalling, G-proteins and ozone-induced processes clearly require further study.

Oxidation of membrane lipids

Degradation of O3 in the apoplast leads to formation of various ROS. Among these, H2O2 is not very toxic to plants cells, which are capable of detoxifying substantial amounts of it. However, formation of hydroxyl radicals in the apoplast from H2O2 and superoxide (O2·−) can initiate lipid peroxidation that results in lipid hydroperoxide (LOOH) formation (Schraudner, Langbartels & Sandermann 1997). Various lipid hydroperoxides and other lipid-based signalling molecules, such as jasmonates are biologically active substances that control a variety of downstream processes. Thus formation of these plasma membrane lipid-based molecules as a result of O3 action in the apoplast may also be regarded as O3-perception.

Intracellular ROS perception

Intracellular ROS are not likely to be sensed through a direct receptor–ligand interaction but instead, their accumulation alters the redox balance of the cell (Lamb & Dixon 1997; Vranováet al. 2002). This can directly affect the activity of transcription factors, second messengers, or enzymes involved in biochemical pathways. Alternatively the cellular redox status can be sensed by the abundant redox-sensitive molecules thioredoxin and glutathione, which in turn transmit the signal forward (Vranováet al. 2002). The ultimate targets in all these cases are either the thiol groups of cysteine residues or the iron–sulphur clusters in the catalytic centres of enzymes. The effect of O3 on the general cellular redox balance can lead to similar responses and represent a way for the cells to respond and acclimate to O3 when the level of ROS from O3 breakdown does not exceed the apoplastic antioxidative capacity, but nevertheless, requires adjustment of defences and metabolism.

The few results available on the O3-perception and immediate downstream signalling support the notion that the action of O3 in plants is due to processes perceived, transduced, and induced by the network of regulatory mechanisms in the cells affected. This seems to involve at least three separate signalling cascades downstream of the perception of O3, since the involvement of the Gα subunit in the O3-lesion formation (Booker et al. 2004; Joo et al. 2005), Gβ or Gβγ signalling to the chloroplast (Joo et al. 2005), and the activation of MAP kinases by O3 (Samuel & Ellis 2002; Ahlfors et al. 2004b; Joo et al. 2005) appear to represent three independent branches of events downstream of O3 perception. One of the major challenges of research on plant O3 responses will be the identification of the signalling processes involved and elucidation of their interactions that result (or do not result in sensitive individuals) in the acclimation of the plant to the external challenge by O3.


Perception of ROS formed by the breakdown of O3 in the apoplast induces an endogenous, active, self-propagating ROS generation when ROS formation from O3 exceeds the apoplastic antioxidative capacity. This active oxidative burst, which continues after the end of the O3 exposure (Fig. 1), is similar to the one observed in the HR (Schraudner et al. 1998; Rao & Davis 1999; Overmyer et al. 2000) and is an integral factor in programmed cell death (PCD). The involvement of PCD in O3-lesion formation is widely accepted (Beers & McDowell 2001; Rao & Davis 2001; Berger 2002), although the morphological and biochemical hallmarks of PCD have been demonstrated only quite recently in tobacco (Pasqualini et al. 2003) and Arabidopsis (Overmyer et al. 2005).

Figure 1.

Temporal changes in ozone-induced activation or accumulation of signalling intermediates. The relative changes of different signalling intermediates are indicated during a typical experiment where plants are exposed to 4–6 h of O3 at a concentration sufficient to induce visible damage in the sensitive accession. Dashed line: tolerant genotypes, solid line: sensitive genotypes.

Similarities between the HR and O3-lesion formation are apparent. The O3-sensitive tobacco cultivar Bel W3 exhibited a similar strong biphasic oxidative burst in response to O3 as during HR, but the tolerant cultivar Bel B had only a modest rise in the first phase of the endogenous radical production (Schraudner et al. 1998). Similar biphasic (Fig. 1), active ROS production has been seen in ozone-sensitive Arabidopsis, birch (Betula pendula), tomato, Malva, and Rumex genotypes, whereas the tolerant counterparts only exhibited the early burst of ROS formation (Pellinen et al. 2002; Wohlgemuth et al. 2002). Consequently, ozone has been recognized as an abiotic elicitor of plant defence responses (Kangasjärvi et al. 1994; Sharma et al. 1996; Sandermann et al. 1998; Rao et al. 2000a), which also makes it a good tool to study, without physically touching the plant, signalling cascades that involve apoplastic ROS formation in the regulation of gene expression and cell death.

ROS production in the chloroplasts has emerged as an important mechanism involved in the acclimation of plants to various abiotic stresses (Karpinski et al. 1999; Mullineaux & Karpinski 2002). Since ROS signalling from chloroplasts is central to both light and wound stress signalling (Fryer et al. 2003; Chang et al. 2004), there is no reason to believe that chloroplasts would not be involved in plant O3 responses, or in any other related cellular process that involves ROS or redox signalling. Accordingly, it was shown that in Arabidopsis the O3-induced ROS production started first in the chloroplasts of the stomatal guard cells, and spread later to the adjoining cells (Joo et al. 2005). DCMU, an inhibitor of chloroplast electron transport, suppressed the early chloroplastic ROS production but did not interfere with ROS production in the cytoplasm and membranes of guard cells. The late peak of ROS production in cells adjacent to the guard cells was also largely suppressed by DCMU. The early chloroplastic oxidative burst was a result of signalling through the heterotrimeric G-protein (or the Gβγ complex) and was distinguishable from the Gα-mediated activation of membrane-bound NADPH oxidases, which was necessary for intercellular signalling during lesion spread and cell death. The observation that the oxidative burst was activated in guard cell chloroplasts even when the NADPH oxidases were inhibited or absent implies that communication of the oxidative stress signal to chloroplasts does not depend on activation of these membrane-associated oxidases, but is a result of signal produced in the apoplast that is perceived in the chloroplast.

A direct connection of chloroplasts with plant O3 responses and acclimation to changed redox status is also provided by the rapid and co-ordinated down-regulation of many nuclear genes encoding chloroplast proteins, presumably as a result of chloroplast-derived signals (Mahalingam et al. 2005), and also by the O3-sensitive Arabidopsis lcd1/rcd2 mutant (described later), which is deficient in a transmembrane chloroplast envelope protein (Barth & Conklin 2003). However, the details on the involvement of chloroplasts in plant O3 responses and the role of the apparent communication between the chloroplasts and other subcellular components in O3-related processes remain to be elucidated.

A superoxide-generating NADPH oxidase has long been suspected to be a prominent source of ROS during the oxidative burst in the HR. Accordingly, DPI, an inhibitor of flavin-containing oxidases, reduced the extent of ozone symptom formation in Arabidopsis, tomato and Malva sylvestris (Rao & Davis 1999; Overmyer et al. 2000; Wohlgemuth et al. 2002). Even though DPI is regarded as an inhibitor of the NADPH oxidase, it also affected the O3-induced H2O2 production in birch (Betula pendula), tomato and tobacco (Pellinen, Palva & Kangasjärvi 1999; Wohlgemuth et al. 2002), which is not surprising since superoxide will be dismutated to H2O2 either spontaneously, or by the action of SOD. The activation of the NADPH oxidase by O3 (or ROS from O3 degradation) was dependent on G-proteins (Joo et al. 2005). The first peak of the bi-phasic oxidative burst elicited by O3 in wild-type Arabidopsis plants was almost entirely missing in mutants of G-protein α and β subunits. The late ROS production in the cells next to guard cells was normal in plants lacking the Gβ protein, but was missing in plants lacking the Gα protein, which means that the ROS accumulation in the adjoining cells was triggered by extracellular ROS produced by guard cell NADPH oxidases, encoded by the ATRBOHD and ATRBOHF genes (Joo et al. 2005). Similarly in tobacco, O3 activated the NtrbohF gene encoding a NADPH oxidase (Langebartels et al. 2002b). It has also been shown that H2O2 accumulation in tobacco apoplast after O3 exposure is a result of dismutation of NADPH oxidase-generated superoxide to H2O2 (Langebartels et al. 2002b). All these results support the importance of the NADPH oxidase in the O3-induced oxidative burst. However, the role of the ROS produced by NADPH oxidase seems to be more important for the cell-to-cell signalling responsible for the induction of ROS production in the adjoining cells from multiple subcellular sources, which in turn seems to be more important for the actual cell death process (Joo et al. 2005).

In addition to NADPH oxidases, cell wall peroxidases and oxalate oxidases have also been proposed as possible ROS sources (Lamb & Dixon 1997; Bolwell et al. 2002). In addition to the surface of the plasma membrane where the NADPH oxidase activity is expected, the early O3-induced H2O2 accumulation was also observed in the cell wall of birch (Betula pendula) exposed to ozone (Pellinen et al. 1999). This suggests that two distinct O3-induced sources for H2O2 generation were operational. Furthermore, during the late phases of O3-induced cell death in birch, H2O2 production occurred also in the cytoplasm, peroxisomes and mitochondria (Pellinen et al. 1999). This was also evident in Arabidopsis, in which the late, tissue damage-associated component of the oxidative burst arose from multiple cellular sources (Joo et al. 2005). The significance of the O3-induced ROS accumulation in both apoplastic and symplastic cellular compartments has also been evident in long-term field experiments with elevated O3: it has been shown that O3-induced cellular H2O2 production takes place in the O3-sensitive accessions of both aspen (Populus tremuloides) and birch (Betula pendula) (Oksanen et al. 2003). Altogether, these results indicate that a multitude of ROS-generating systems are involved in the O3-triggered ROS production in both controlled experiments as well as in the field-grown plants.


Mitogen-activated protein kinase (MAPK) cascades are a conserved signal transduction system in all eukaryotes and their importance for plants is also well known (Jonak et al. 2002; Baier et al. 2005). The universal structure of this signalling module consists of a MAP kinase kinase kinase (MAPKKK) that phosphorylates a MAP kinase kinase (MAPKK) that in turn phosphorylates a MAP kinase (MAPK). Of the 20 MAPKs in the Arabidopsis genome (MAPK Group 2002), the two primary oxidative stress-related are AtMPK6 and AtMPK3. They, and their orthologues in tobacco (SIPK and WIPK, respectively), are induced, among other stresses, by ozone (Samuel, Miles & Ellis 2000; Samuel & Ellis 2002; Ahlfors et al. 2004b; Joo et al. 2005). AtMPK6/SIPK and AtMPK3/WIPK are also activated by hydrogen peroxide and superoxide (Kovtun et al. 2000; Samuel et al. 2000; Moon et al. 2003). In addition to being activated by ROS, the activation of Arabidopsis AtMPK6 and AtMPK3 by constitutively active forms of the upstream MAPKKs AtMKK4 and AtMKK5 induced endogenous hydrogen peroxide production and cell death (Ren, Yang & Zhang 2002).

Although both AtMPK6 and AtMPK3 are rapidly activated by O3 (Fig. 1), the regulation of their activity by O3 is different. AtMPK3 was up-regulated by ozone on transcriptional, translational as well as on post-translational levels, whereas only post-translational activation of the kinase activity of AtMPK6 was detected (Ahlfors et al. 2004b). In addition, the activation of AtMPK3 lasted longer than that of AtMPK6. Plant ozone sensitivity and the expression of antioxidant genes is affected by these kinase classes, since both suppression and overexpression of the tobacco SIPK (the AtMPK6 orthologue) led to increased ozone sensitivity and changes in the expression of APX and GST (Samuel & Ellis 2002). Interestingly, at least in tobacco, SIPK also seems to regulate the activity of WIPK (the AtMPK3 orthologue), since in the SIPK overexpression lines, WIPK activity induced by ozone was significantly reduced, whereas the opposite was true for the SIPK suppression line (Samuel & Ellis 2002).

Activation of MAP kinases by phosphorylation generally leads to nuclear localization of, and activation of transcription factors by the MAPK. Accordingly, the phosphorylated AtMPK3 and AtMPK6 were translocated rapidly to the nucleus within 30 min of initiating O3 exposure (Ahlfors et al. 2004b). The role and function of these MAP kinases in O3-exposed plants is still, however, unknown, although there is recent evidence that they might be connected to the hormonal responses, as described below.


Plant hormones play key roles in ozone lesion development. ABA, salicylic acid (SA), ethylene, and jasmonic acid (JA) are important in determining the degree of plant ozone sensitivity and O3-lesion initiation, propagation and containment (Overmyer, Brosché & Kangasjärvi 2003). The role of ABA in the stomatal regulation and O3 influx has been discussed above. Ethylene and salicylic acid are needed for the development of the visible O3-lesions and jasmonic acid acts to limit and contain the lesion spread. This is also illustrated by the fact that the mutants and accessions of Arabidopsis and other species, first described as O3-sensitive, have turned out to be either partially JA insensitive (Arabidopsis mutants oji1, rcd1, ecotype Cvi-0, poplar clone NE-388), or ethylene over-producers (Arabidopsis mutants rcd1, oji1 and ecotypes Ws and Kas-1) (Koch et al. 2000; Overmyer et al. 2000; Rao et al. 2000b; Kanna et al. 2003; Tamaoki et al. 2003a).

The three processes that determine ozone lesion formation after stomatal control, apoplastic antioxidants, and ROS perception – initiation, propagation and containment of the O3-lesion – can be depicted as a self-amplifying loop termed oxidative cell death cycle (Van Camp, Van Montagu & Inzé 1998; Overmyer et al. 2000, 2003; Fig. 2). In this cycle the endogenous, cell death-driving ROS production triggered by O3 is ethylene-dependent (Overmyer et al. 2000; Moeder et al. 2002; Wohlgemuth et al. 2002; Kanna et al. 2003). ROS production drives the SA-dependent (Örvar, McPherson & Ellis 1997; Rao & Davis 1999) cell death, which continues until the third, JA-dependent component, antagonistic to lesion propagation, contains the enlargement of the lesion.

Figure 2.

The oxidative cell death cycle. Ozone-derived radicals induce endogenous reactive oxygen species (ROS) production (1) which results in salicylic acid (SA) accumulation and programmed cell death (2). Cell death triggers ethylene (ET) production, which is required for the continuing ROS production responsible for the propagation of cell death (3). Jasmonates counteract the progression of the cycle by antagonizing the cell death promoting function of SA and ethylene (4). Abscisic acid (ABA) antagonizes ethylene function in many situations and might also have this role in ozone-induced cell death (5). Mutually antagonistic interactions between ethylene, SA and jasmonic acid (JA) are indicated with red bars.

MAP kinases are connected to both ROS and cell death (Kovtun et al. 2000; Ren et al. 2002) and MAPK involvement in ethylene, SA, and ABA signalling has been established in different contexts (Zhang & Klessig 1997; Kim et al. 2003; Ouaked et al. 2003; Xiong & Yang 2003). The question of, whether plant hormones regulate MAP kinase signalling in ozone-induced cell death, or vice versa, has been addressed by Ahlfors et al. (2004b), who showed that the initial activation of AtMPK6 and AtMPK3 by ozone was independent or upstream of SA, ethylene or JA signalling since the activation pattern was similar in mutants deficient in the respective cascades. However, hormones are intertwined with MAPKs since the activation of AtMPK6 was prolonged and the AtMPK3 activation delayed in the ethylene insensitive etr1 mutant. Additionally, the basal expression level of AtMPK3 was only half of the wild-type levels in salicylic acid-insensitive and -deficient accessions, and this lower level of expression was also reflected in AtMPK3 activity. These results further support the notion that O3-induced hormone synthesis and signalling are connected with MAP kinase cascades.

Initiation of O3 lesion

Ozone has been shown to induce the death of a small number of cells, visible at the microscopic level, also in resistant plants (Overmyer et al. 2000). Similarly, when tobacco plants were exposed to O3 in consecutive days, the number of lesions was determined during the first day and in the subsequent exposures the existing lesions simply expanded without initiation of new lesion (Wohlgemuth et al. 2002). The O3 lesions are not randomly distributed throughout the leaf, but are preferentially located in the close proximity of the second and third degree veins (Schraudner et al. 1998; Wohlgemuth et al. 2002). In the systemic acquired resistance (SAR), an analogous ROS-dependent death of a small number of cells close to the veins, termed micro-HR, is essential for the development of SAR (Alvarez et al. 1998). In the systemic leaves, cell death as a response to a transmissible signal from the cells in contact with the pathogen does not spread further from these clusters of cells. This is in contrast to the pathogen-infected ‘primary’ leaves in which cells around the primary infection site die by PCD, as a result of unidentified signals from the cell death initiation site. This raises the question whether the formation of O3 lesions uses the same process and if the small number of dead cells in the leaves of O3-tolerant plants is analogous to the micro-HR in the systemic leaves, and whether the enlargement of the lesion in the O3-sensitive plants is analogous to the spreading of the HR lesion in the primary leaves of pathogen-infected plants.

In plants, SA is best known for its role in SAR (Raskin 1995; Hoeberichts & Woltering 2002) for which it seems to be essential. It is also required for the execution of HR-like cell death (Durner, Shah & Klessig 1997). Ozone exposure and pathogen attack both induce SA synthesis within a few hours after the beginning of the exposure (Örvar et al. 1997; Overmyer et al. 2005; Fig. 1). The role of SA has mostly been demonstrated through experiments with transgenic, SA-degrading NahG plants and the SA-insensitive Arabidopsis mutant npr1. These studies have shown that O3-induced cell death is abolished in the absence of SA or its action (Örvar et al. 1997; Rao & Davis 1999; Overmyer et al. 2005). The integral role for SA in cell death is also supported by experiments in which exogenously applied SA significantly increased the ozone sensitivity of otherwise tolerant genotypes (Rao et al. 2000a; Mazel & Levine 2001). Furthermore, double mutant analysis in Arabidopsis has shown that O3-sensitive accessions became tolerant when either the transgene NahG or the npr1 mutation was introduced to the sensitive background (Örvar et al. 1997; Rao et al. 2000a; Overmyer et al. 2005).

All this suggests that SA has a vital role in cell death and that without SA active PCD is not initiated in O3-exposed plants. However, Rao & Davis (1999) have shown that inhibition of SA accumulation in O3-exposed plants resulted in cell death either by necrosis (as opposed to PCD), or by HR-like programmed cell death (PCD), depending on the duration of exposure to O3. In short-term O3 exposures the SA-deficient NahG Arabidopsis plants were more tolerant to O3 than the wild-type plants, whereas in long-term exposures NahG plants developed necrotic lesions without the involvement of the active superoxide formation typical of the lesions in short-duration O3 exposures. The necrotic cell death was interpreted as a result of a gradual depletion of antioxidative capacity, which led to a drastic shift in the cellular redox balance and eventually to cell death. Thus, SA may also have a role in the up-regulation of antioxidative systems during lesion spread and also have a protective function when the SA concentration increases (Fig. 1) during lesion formation.

It is also possible that under specific conditions, O3– or more likely the ROS generated directly from O3 degradation – can also physically damage cellular components and cause cell death. Hence, it has been suggested that death by both rampant oxidation and PCD may occur depending on the O3 concentration (Pell et al. 1997). This means that the possibility for mosaics of PCD and necrotic cells occurring in the same O3-exposed tissue cannot be excluded. Consequently, signals from the cells that have undergone necrotic cell death caused by the ROS directly formed from O3 may trigger surrounding cells to die by PCD. This results in the beginning of lesion propagation and the formation of the visible lesion.

Lesion propagation, as discussed below, is clearly an ethylene-dependent process, but it has been suggested by Rao, Lee & Davis (2002) that the O3-induced ethylene synthesis could be SA-dependent. However, the results presented did not answer whether the lower ethylene evolution in O3-exposed NahG or npr1 plants was a result of direct regulatory action of SA on ethylene synthesis, or a result of less initial cell death due to lack of SA. Thus, whether, and how, SA is involved in the regulation of ethylene biosynthesis can still be regarded as an open question until it is known whether the activation of ethylene biosynthesis is dependent on initial cell death and the primary signals responsible for the up-regulation of the rapidly appearing ethylene biosynthesis have been identified.

Lesion propagation

Stress ethylene production is intimately linked to ozone damage in several species (Tingey, Standley & Field 1976; Mehlhorn & Wellburn 1987; Overmyer et al. 2000; Rao et al. 2002) and it has been shown that lesion propagation in O3 damage is under the control of ethylene (Overmyer et al. 2000; Rao et al. 2000a, 2002; Moeder et al. 2002; Kanna et al. 2003; Tuominen et al. 2004). Ethylene production is one of the fastest responses of plants to ozone (Tuomainen et al. 1997; Vahala, Schlagnhaufer & Pell 1998; Overmyer et al. 2000; Fig. 1) and is well-correlated with the extent of cell death (Langebartels et al. 1991; Tuomainen et al. 1997; Tamaoki et al. 2003a). The ethylene insensitive Arabidopsis mutants (etr1, ein2, ein3) are O3 tolerant. Accordingly, also the transgenic birch (Betula pendula) trees expressing Arabidopsis ethylene receptor gene with the dominant etr1-1 mutation, in addition to being ethylene insensitive, were also more tolerant to O3 than the background clone (Vahala et al. 2003). In ethylene overproducing Arabidopsis mutants (eto1, eto2, eto3), lesion propagation is aggravated by the increased ethylene synthesis. Accordingly, a role in promoting cell death has been assigned to ethylene in ROS-dependent PCD (de Jong et al. 2002; Overmyer et al. 2003).

Ethylene biosynthesis is regulated at the level of the production of its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC), synthesized by ACC synthases (ACS) from S-adenosyl-methionine. The conversion of ACC to ethylene by ACC oxidases is considered less rigorously controlled (Wang, Li & Ecker 2002). Ozone-induced ethylene biosynthesis is the result of induction of specific members of ACS gene family, which consists of several members that show differential expression patterns in plant organs during development and as a response to external challenges. In Arabidopsis, a specific member of the gene family, AT-ACS6, is rapidly induced by ozone (Vahala et al. 1998; Overmyer et al. 2000; Tamaoki et al. 2003a). Similarly in tomato, a bi-phasic, sequential induction of ACS genes by ozone has been observed (Nakajima et al. 2001; Moeder et al. 2002): first the LE-ACS1B and LE-ACS6 genes were up-regulated by ozone very rapidly, followed by a sharp reduction in their mRNA levels and a simultaneous induction of LE-ACS2.

The silencing of LE-ACS2 with anti sense technique leads to an almost complete block of ethylene synthesis in developing tomato fruit (Oeller et al. 1991). In the leaves of O3-exposed LE-ACS2 anti sense plants, however, ethylene biosynthesis increased rapidly during the initial phase (LE-ACS1B and LE-ACS6 induction), but the increase in ethylene evolution during the second phase, when LE-ACS2 induction takes place in the wild-type plants, did not occur (Moeder et al. 2002). Accordingly, O3-sensitivity of the LE-ACS2 anti sense plants did not differ from the untransformed control. On the contrary, when the tomato LE-ACS6 was transformed to tobacco in anti sense orientation, low initial rates of O3-induced ethylene production were observed in the transgenic plants and the extent of O3 damage was lower than in the nontransformed controls (Nakajima et al. 2002). This suggests that the enzymes encoded by the sequentially induced members of the ACS gene family may have a distinct function in the process and that the first phase of ethylene synthesis was sufficient to trigger the ethylene-dependent processes that resulted in O3-lesion formation.

The very early activation of ethylene biosynthesis before the activation of ACC synthase gene expression is suggested to be regulated at the post-translational level. It has been shown that in tomato the stability of LE-ACS2 protein is regulated by phosphorylation of a Ser-460 residue in the C-terminal part of the enzyme (Tatsuki & Mori 2001), which presumably affects the binding of the ETO1 protein to the ACS dimer and prevents the degradation of the active dimer by the 26S proteasome (Wang et al. 2004). Kim et al. (2003) suggested the involvement of SIPK (AtMPK6 homologue) in the activation of ethylene biosynthesis, and the down-regulation of this kinase by the ethylene-dependent feedback loop in tobacco. Additionally, Liu & Zhang (2004) demonstrated that Arabidopsis MPK6 directly phosphorylates AT-ACS6 protein, which prevents its proteolytic degradation and causes the up-regulation of ethylene synthesis. This model is in accordance with the prolonged AtMPK6 activity in O3-exposed ethylene-insensitive Arabidopsis etr1 mutant (Ahlfors et al. 2004b), with the faster activation of AtMPK6 (Overmyer et al. 2005) as well as higher ethylene biosynthesis and AT-ACS6 induction (Overmyer et al. 2000) in the O3-sensitive rcd1 mutant. Similarly, inhibition of protein kinase activity with the general kinase inhibitor K252a prevented the O3-induced increase in ACC synthase activity in tomato (Tuomainen et al. 1997) and also O3 damage in the O3-sensitive Arabidopsis rcd1 mutant (Overmyer et al. 2005). It has also been shown that inhibition of protein phosphatase activity with calyculin A significantly increased ethylene evolution and ACC synthase activity in the absence of other external elicitation (Spanu et al. 1994; Tuomainen et al. 1997). This treatment also significantly increased cell death in the O3-sensitive rcd1 mutant (Overmyer et al. 2005). Whether the AtMPK6-dependent phosphorylation of AT-ACS6 (Liu & Zhang 2004) is involved in the activation of O3-induced ethylene synthesis as a result of increased cell death, or vice versa, or whether these two processes are independent requires further study.

Lesion containment

Without a counteracting regulatory system that limits the spread of the lesion once it is initiated, induction of spreading cell death would result in a progressive destruction of the whole organ. This is evident, for example, in the Arabidopsis spontaneous lesion mutant lsd1 (Jabs, Dietrich & Dangl 1996), where a ‘runaway cell death’ spreads throughout the whole leaf once the lesion is initiated. Two different hormonal mechanisms can be suggested to be responsible for the containment of lesion spread.

Ethylene itself can be responsible for the containment of (the ethylene-dependent) O3 lesion propagation since ethylene causes desensitization of the cells to its own action (Wang et al. 2002). The ethylene receptor acts as a suppressor of ethylene signalling when it is not in contact with the hormone and this suppression is released after ethylene binds to the receptor. It is thought that the ethylene-induced synthesis of new, unoccupied receptor molecules is responsible for the desensitization of ethylene signalling. Up-regulation of genes encoding ethylene receptors have also been seen in ozone-exposed tomato (Moeder et al. 2002). Thus the O3-induced synthesis of new ethylene receptor proteins could, in part, lead to decreased ethylene sensitivity and down-regulation of ethylene-dependent lesion spread. Whether the up-regulation of new receptor synthesis takes place in, or around, the developing lesion is, however, unknown.

Other hormones also act in lesion containment. Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are the most studied of the linolenic acid-derived signalling molecules in plants that are collectively referred to as oxylipins or jasmonates (Farmer, Weber &Vollenweider 1998). Exposure to ozone stimulates jasmonate biosynthesis in plants (Rao et al. 2000a; Tuominen et al. 2004; Fig. 1). In the oxidative cell death cycle, jasmonates protect tissues from ROS-induced cell death and thus counteract the effects of SA and ethylene (Fig. 2). Örvar et al. (1997) showed that pretreating tobacco plants with jasmonates inhibited ozone-induced cell death. The same was evident in Arabidopsis, in which jasmonate treatment also reduced the amount of SA produced in response to ozone (Rao et al. 2000b), jasmonate-insensitive and -deficient Arabidopsis mutants jar1, coi1, fad3/fad7/fad8, and oji1 were hypersensitive to ozone (Overmyer et al. 2000; Rao et al. 2000b; Kanna et al. 2003; Tuominen et al. 2004), and MeJA applied after ozone halted lesion spread in the ozone-sensitive rcd1 mutant and Ws ecotype (Overmyer et al. 2000; Kanna et al. 2003). Not all JA-insensitive mutants are, however, sensitive to O3. The jasmonate insensitive jin1 is tolerant to O3 (Nickstadt et al. 2004), which suggests that JA has at least two different roles in O3-responses. Accordingly, it was shown that the MYC-type transcription factor (AtMYC2) encoded by JIN1 is required to discriminate between two different branches of jasmonate-responses (Lorenzo et al. 2004). Both these branches of JA signalling seem to be, however, involved in O3-related processes, one in lesion formation and the other in lesion containment, since lesion formation seems to require AtMYC2 (JIN1) and lesion containment requires JAR1 and COI1.

Interactions between the hormonal signalling cascades

The evidence on the hormonal control of plant O3 responses is strong and the balance between SA, ethylene and JA is very likely to be largely responsible for the processes involved (Overmyer et al. 2003; Tamaoki et al. 2003b; Tuominen et al. 2004). The balance seems to be accomplished by mutual interactions. Since JA prevented the O3-induced accumulation of SA (Örvar et al. 1997), it seems that JA's antagonism to cell death is mediated at least partly through its effect on SA. JA also antagonizes ethylene signalling. This interaction is mutually antagonistic, since ethylene also inhibited JA-induced gene expression (Tamaoki et al. 2003b; Tuominen et al. 2004). Because ethylene levels in ozone-treated JA-insensitive jar1 mutant were similar to wild type (Overmyer et al. 2000) and MeJA application did not reduce ethylene emission in eto1 (Tuominen et al. 2004), JA antagonism to ethylene is not likely to take place at the level of biosynthesis. Results of triple response assays suggested that JA action on ethylene was downstream of biosynthesis but upstream of CTR1, a raf-type protein kinase active in ethylene signalling just downstream of the receptor (Tuominen et al. 2004). Thus, JA could affect ethylene signalling at the receptor level by decreasing ethylene sensitivity in a receptor-dependent manner. Accordingly, JA-induced up-regulation of an ethylene receptor isoform has been discovered in micro-array studies (Schenk et al. 2000).

In birch (Betula pendula) and Arabidopsis, both ethylene and JA accumulation were high in the O3-sensitive accessions, whereas in the O3-tolerant accessions JA concentration did not increase (Vahala et al. 2003; Tuominen et al. 2004; Fig. 1). The suggested involvement of JA in lesion containment may at first seem contradictory to JA accumulation in the sensitive accessions. However, because JA biosynthesis appears to be limited by substrate availability (Laudert, Schaller & Weiler 2000; Ziegler, Keinänen & Baldwin 2001), it is also possible that the JA accumulation is a consequence of cell death: the substrate for JA synthesis (α-linolenic acid or 13-(S)-hydroperoxylinolenic acid) could originate from membranes in the dying cells, thus resulting in increased JA synthesis, which then halts the ET-dependent lesion propagation by decreasing ethylene sensitivity of the cells. At the tissue level, the balance probably shifts temporally and spatially so that in the first cells the SA- and ET-driven processes prevail, but further away from the site of initiation the JA pathways become progressively more induced, overcome the first processes and containment of cell death follows.


The use of forward and reverse genetics in Arabidopsis has increased our understanding of several basic biological processes and has enabled identification of several new protein classes and revealed their function in sometimes surprising connections. Three separate mutant screens for increased O3 sensitivity have been performed. Some of the mutants found are described below and also in the context of the biological process identified.

vtc1 (soz1). The Arabidopsis ascorbate biosynthesis mutant vtc1 (vitamin C defective 1) was originally isolated in a mutant screen for increased ozone sensitivity and originally called soz1 (sensitive to ozone 1). Identification of SOZ1 revealed that the gene encoded a GDP-mannose pyrophosphorylase involved in ascorbate biosynthesis (Conklin et al. 1996, 1999). Accordingly, soz1 was renamed as vtc1. Its significance in plant stress responses is reviewed in Conklin & Barth (2004) and thus not treated here in more detail. Identification of soz1 as a component of ascorbate biosynthesis indicates the important role and contribution of antioxidative systems in plant oxidative stress tolerance. However, the mechanisms and exact role of chloroplastic, cytosolic or apoplastic ascorbate in plant O3-responses remains to be elucidated further.

soz2 (lcd1-1 rcd2) is a second mutant from the same mutant screening series where vtc1 was discovered (Barth & Conklin 2003). The phenotype of soz2 under moderate light levels (120 µmol m−2 s−1) is pale when compared to the wild type, but the O3 damage is separate from the reticulate phenotype and appears as HR-like cell death. Microscopic analysis revealed that in moderate light-grown, non-O3-treated soz2 plants, the cell number and density in palisade parenchyma was lower than in wild type and the pale phenotype of the mutant was a result of the lower cell density, not lower chlorophyll content or chloroplast number per cell. Consequently, based on these observations soz2 was renamed lcd1 (lower cell density 1) and it was speculated (Barth & Conklin 2003) that the protein encoded by LCD1 (At2g37860, annotated as expressed protein) could play a role in normal leaf development.

A second allele of lcd1 (lcd1-2) with an identical phenotype was also identified as an O3-sensitive mutant in a separate mutant screen for O3-sensitivity in the authors’ laboratory and named rcd2 (radical-induced cell death 2; compare to rcd1 below). The experiments with rcd2 indicate that when lcd1-2/rcd2 is grown under extremely low light level (below 30 µmol m−2 s−1), the mutant and the wild type are indistinguishable and the lcd1-2/rcd2 does not have the reticulate phenotype (Kollist et al. in preparation). Furthermore, the cell number and density are the same in the mutant and the wild type. When the very low light-grown lcd1-2/rcd2 plants are transferred to moderate light (around 150 µmol m−2 s−1), photobleaching occurs in the mutant, without affecting the already-determined cell number and density. Thus the low cell density in lcd1 (Barth & Conklin 2003) seems to be a result of light intensity-dependent processes most likely affecting chloroplastic functions, which results in lower cell density during leaf development. Thus, the low cell density in lcd1 could be regarded as a secondary effect.

LCD1 belongs to a two-member gene family. The second member of the family, At5g22790, encodes a protein with 76% identity and 86% similarity with LCD1. Both proteins have an unknown function and are predicted to be targeted to chloroplasts. Indeed, proteomic analysis of chloroplast envelope proteins (Ferro et al. 2003) indicated that At5g22790 is located in the chloroplast envelope membrane. Both proteins have two C-terminal transmembrane domains and the mutation in both lcd1-1 and rcd2 (lcd1-2) is located in the second C-terminal transmembrane domain – in lcd1-1 the last amino acid is missing and in rcd2 (lcd1-2) a mutation in an intron splicing site results in a truncated protein lacking the second transmembrane domain. Thus, the C-terminal part of the protein seems to be required for its proper function.

How the putative chloroplast envelope protein relates to acclimation to the ROS produced from, and by O3 in the apoplastic space is unknown, but the lcd1/rcd2 suggests that chloroplastic processes are involved. It could be speculated that the transmembrane chloroplast envelope membrane protein LCD1/RCD2 could be involved in the communication between chloroplasts and extrachloroplastic subcellular compartments, especially since the gene has been identified in two separate mutant screens for increased O3-sensitivity and chloroplasts have been shown as a very early target for a signal from the apoplast of O3-exposed plants (Joo et al. 2005; Mahalingam et al. 2005). When exposed to O3, lcd1/rcd2 has significantly increased superoxide production associated with the developing O3 lesion (Kollist et al. in preparation). In clean air, however, under moderate light levels (150 µmol m−2 s−1) the lcd1/rcd2 mutant displays constant H2O2 accumulation around the veins in a pattern and manner similar to the H2O2 accumulation during acclimation to high light (> 1000 µmol m−2 s−1) conditions (Fryer et al. 2003), a process that involves a redox signal-mediated communication between the chloroplast and the nucleus.

rcd1 was isolated in a screen for increased O3-sensitivity. In addition to O3-sensitivity, rcd1 has several other phenotypes related to growth, development and hormone biology (Overmyer et al. 2000; Ahlfors et al. 2004a). A second allele of rcd1 was identified in a mutant screen for paraquat tolerance (Fujibe et al. 2004). Thus, rcd1 is sensitive to conditions that lead to the formation of apoplastic ROS, which induces a spreading cell death phenotype in the mutant, but it has increased tolerance to ROS formation in the chloroplast manifested as increased tolerance to the ROS-generating herbicide paraquat (Ahlfors et al. 2004a; Fujibe et al. 2004). rcd1 has also increased tolerance to UV-B (Fujibe et al. 2004). RCD1 has also been reported earlier under the name CEO1 (Clone Eighty One), identified as an Arabidopsis cDNA that complements a yeast mutation that renders yeast sensitive to oxidative stress (Belles-Boix et al. 2000), and ATP8, an Arabidopsis protein that interacts with the movement protein of turnip crinkle virus (Lin & Heaton 2001). The RCD1 protein (At1g32230) contains a WWE domain predicted to be involved in protein–protein interactions (Aravind 2001), nuclear localization sequences, a ‘PARP’ domain (NAD-binding catalytic core for ADP-ribosyl transferases), and an unidentified C-terminal protein–protein interaction domain identified in yeast two-hybrid analysis (Belles-Boix et al. 2000). The C-terminal interaction domain in RCD1/CEO1 was shown to interact with several transcription factors or DNA-binding proteins involved in dehydration or osmotic stress responses, such as DREB2A, which is a central transcription factor in the ABA-independent responses to osmotic stress (Liu et al. 1998). The mutation in RCD1 seems to affect mostly hormone-related processes, since in micro-array analysis with 6400 genes the few genes that had changed basal expression level in clean-air grown rcd1 are involved in either ethylene-, ABA-, or sugar-related processes (Ahlfors et al. 2004a). Furthermore, rcd1 shows slight insensitivity to both ABA and ethylene in some specific responses to these hormones, which is reflected, for example, in higher stomatal conductance in rcd1 than in Col-0 wild type. It was speculated that the function of RCD1 could relate to post-translational modification of its targets by ADP ribosylation, which remains to be determined.

rcd3 was isolated in the same mutant screen as the two other rcd mutants described above. Similar to rcd1, also rcd3 has higher stomatal conductance than the Col-0 wild type. Unlike in rcd1, micro-array analysis with 6400 genes did not reveal any genes with different basal expression between clean air-grown rcd3 and Col-0 (Lamminmäki, Kangasjärvi et al. unpublished), which suggests that the gene mutated might be involved in a process that could be specifically related to O3-responses and the effect of the mutation becomes visible only when the plants are challenged with oxidative stress. The future identification of RCD3 is thus expected to reveal a specific process related to acclimation to O3 or stomatal regulation.

oji1 (ozone-sensitive and jasmonate-insensitive 1) was identified in a screening of a T-DNA insertion mutation population in the ecotype Ws (Kanna et al. 2003). The mutant has severe foliar injury and higher ethylene emission than the Ws wild type under O3. The mutant was also insensitive to jasmonate inhibition of root elongation, and the expression of a commonly used JA marker gene, VSP1 (VEGETATIVE STORAGE PROTEIN 1) and the jasmonate inhibition of O3-damage were reduced in oji1 when compared to Ws. The ascorbate content of oji1 was similar to the wild type, thus the O3-sensitivity of the mutant is attributable to the increased ethylene evolution and/or decreased jasmonate sensitivity. As shown by Tuominen et al. (2004), these two are not necessarily separate since they have a mutually antagonistic interaction with each other. However, oji1 is another example showing the significance and role of both ethylene and jasmonate in plant O3 sensitivity.

The location of the T-DNA tag in oji1 was preliminarily localized with TAIL-PCR (Thermal Asymetric Interlaced Polymerase Chain Reaction) between At3g61810 and At3g61820 (Kanna et al. 2003). The former is a β-1,3, glucanase and the latter is an aspartyl protease family protein with one predicted transmembrane domain and a putative targeting to endomembrane system. It remains to be verified if the OJI1 gene encodes the aspartyl protease protein and what is its function in the jasmonate signalling or regulation of ethylene biosynthesis.

The identification of the mutated genes in the O3-sensitive mutants described above has identified new, and verified already known processes important for plant O3 tolerance. The role of ascorbate is strengthened by these mutants and the (still unclear) role for chloroplasts in plant O3 responses is evident, as demonstrated by the lcd1/rcd2 mutant. The importance of hormonal regulation and especially the role of ethylene in determining plant ozone sensitivity has been verified by the rcd1 and oji1 mutants, and mutant analysis has facilitated the elucidation of the oxidative cell death cycle (Overmyer et al. 2000, 2003), which seems to have universal relevance in the regulation of the spread of cell death, not only as a response to ozone, but in other responses as well in which induced lesion formation takes place. The control of ozone entry to the leaf, namely stomatal conductance seems also to be a central issue, since two O3-sensitive mutants, rcd1 and rcd3 seem to have deficient stomatal regulation resulting in an increased initial flux of O3 to the intercellular air space.


Based on the results reviewed above, the following sequence of events in O3-exposed plants, shown in Fig. 3, can be proposed. The first structure that O3 meets in plants is the stomatal guard cells, which are affected within minutes. Apparently the ROS from O3 breakdown in the apoplast of guard cells induce stomatal closure and/or inhibition of stomatal opening, eventually decreasing stomatal conductance. Under high external O3 concentrations the influx of O3 during the early phase exceeds the capacity of the apoplastic antioxidants to detoxify the ROS formed from O3 degradation. This causes the apoplastic ROS level to increase over a threshold that elicits several downstream responses (numbers 1 and 11 in Fig. 3). At least two separate chains of events are induced. First, O3 induces ROS production in the chloroplasts of the guard cells through the heterotrimeric G-protein (or the Gβγ complex) (numbers 2 and 3). Then, ROS production in the guard cell plasma membrane by  NADPH oxidases is activated in a G-protein α-subunit-dependent way (number 4). Consequently ROS production and initiation of the SA-dependent cell death (number 5) spread to the adjoining cells. MAP kinase cascades are also activated within minutes from the beginning of O3 exposure (number 6). These two early processes are, however, independent from each other, since the early activation of MAP kinases takes place in both O3-sensitive and -resistant accessions and does not require G-protein. Thus, MAP kinases do not appear to be directly involved in the activation of ROS production by NADPH oxidase. However, the activation of AtMPK6 (SIPK in tobacco) is involved in the phosphorylation of ACC synthase, which results in increased ethylene synthesis and (presumably) the autocatalytic, biphasic activation of the genes encoding the O3-induced members of the ACC synthase family. Autocatalytic activation by increasing the stability of ACC synthase by phosphorylation is not, however, the only process by which O3 regulates ethylene synthesis, as greatly increased ethylene synthesis is apparent only in the sensitive accessions (number 7). Either other components are required together with autocatalysis, or the down-regulation of ethylene synthesis is slower or more inefficient in the O3-sensitive accessions.

Figure 3.

Signal transduction and its spatial progression induced by ozone exposure. Ozone is degraded to reactive oxygen species (ROS) in the apoplast (1). Their accumulation is affected by antioxidants such as ascorbate. Apoplastic ROS activate a signal transduction chain depending on heterotrimeric G-proteins (2). The first phase of this chain is dependent on the βγ heterodimer and induces ROS production in the chloroplast (3). The further transmittance of the chloroplastic ROS signal requires the G-protein α-subunit and results in the activation of a plasmamembrane NAPDH oxidase (RBOH), possibly through a small GTPase Rac (4). In parallel with G-proteins, a MAP kinase cascade is activated (6). Although the downstream targets of this cascade are not known, it might have connections to the up-regulation of ethylene (ET) biosynthesis (7). In addition to ethylene, salicylic acid biosynthesis is also induced (5). Calcium influx takes place in the early stages of signal transduction (8) and both the activation of MAP kinases and NADPH oxidase are calcium-dependent. The ROS production by the NADPH oxidase is essential for the spreading of the ozone-induced signal. Again, heterotrimeric G-proteins are involved in receiving the signal and passing it forward in an ethylene-dependent manner (9). When the ozone-induced spreading cell death is halted, jasmonate biosynthesis is up-regulated and this blocks ethylene signalling, most likely at the level of ethylene perception (10). The processes described above regulate a set of ozone-induced responses including cell death and the transcription of defence-related genes (11).

Spread of the O3-induced oxidative burst is activated after the perception of O3 in the lesion initiation site. The signal responsible for the cell-to-cell communication is produced by the NADPH oxidase (number 4), whose activation requires G-protein α subunit. The ROS produced by the NADPH oxidase in the lesion initiation site are perceived by the surrounding cells, in which G-protein-mediated activation of ROS production is induced in several subcellular compartments. Either the spread of the signal to the neighbouring cells, or the competence of these cells to perceive the ROS signal, or to respond to it seems to be ethylene-dependent (numbers 7 and 9). This process repeats in the ‘oxidative cell death cycle’, which results in the formation of the visible O3 lesion. The oxidative burst differs in O3-tolerant and -sensitive accessions: both have an initial, fast burst of ROS as a response to O3, but only in the sensitive accessions a second, ethylene- and G-protein-dependent burst that is required for the spreading cell death is apparent (Fig. 1).

Cell death during the lesion spread creates an increasing concentration of membrane lipid peroxidation products, which serve as a substrate for jasmonate biosynthesis. Accordingly, only in the sensitive accessions is high JA accumulation observed. Short-distance signalling by increasing JA accumulation decreases ethylene sensitivity in the cells in the perimeter of the spreading lesion, resulting in a gradual decrease of (the ethylene-dependent) ROS production and consequently containment of cell death (Fig. 3, number 10).