Gene Networks in Plant Ozone Stress Response and Tolerance


  • Agnieszka Ludwikow,

    1. Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznan, Poland
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  • Jan Sadowski

    Corresponding author
    1. Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznan, Poland
    2. Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, Poland
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*Author for correspondence.
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For many plant species ozone stress has become much more severe in the last decade. The accumulating evidence for the significant effects of ozone pollutant on crop and forest yield situate ozone as one of the most important environmental stress factors that limits plant productivity worldwide. Today, transcriptomic approaches seem to give the best coverage of genome level responses. Therefore, microarray serves as an invaluable tool for global gene expression analyses, unravelling new information about gene pathways, in-species and cross-species gene expression comparison, and for the characterization of unknown relationships between genes. In this review we summarize the recent progress in the transcriptomics of ozone to demonstrate the benefits that can be harvested from the application of integrative and systematic analytical approaches to study ozone stress response. We focused our consideration on microarray analyses identifying gene networks responsible for response and tolerance to elevated ozone concentration. From these analyses it is now possible to notice how plant ozone defense responses depend on the interplay between many complex signaling pathways and metabolite signals.

In the past few years evidence has come to light about stratospheric ozone depletion and an increase of tropospheric ozone concentration (Overmyer et al. 2000; Evans et al. 2005). As much as we need ozone in the stratosphere to protect us from harmful UV radiation, ground-level (tropospheric) ozone can be considered as an atmospheric pollutant with direct toxic effects on the terrestrial biosphere obviously affecting plant growth and development (Sandermann 2000; Tausz et al. 2007). Although environmental ozone concentrations are relatively low for ozone stress response and tolerance studies, acute doses are developed in order to understand the mechanism of O3 action on plants (Rao et al. 2000a, 2000b; Vahala et al. 2003; Ludwikow et al. 2004; Kangasjarvi et al. 2005).

Plants respond and adapt to O3 stress to survive under stress conditions at physiological and biochemical levels (Rao et al. 2000a, 2000b; Kangasjarvi et al. 2005; Wittig et al. 2007). The physiological, stomatal and ultrastructural responses to ozone were studied extensively in a low and acute stress chamber experiment as well as in field experiments (Vahisalu et al. 2008). These studies revealed that ozone affects plant growth and development through the induction of oxidative stress and activation of programmed cell death. Ozone enters the plant cell through the stomata and breaks down in the apoplast to produce different reactive oxygen species (ROS) (Overmyer et al. 2003; Baier et al. 2005; Foyer and Noctor 2005; Kangasjarvi et al. 2005). Intracellular ROS production elicits a sequence of cellular events resulting in the adjustment of defense, metabolism and signaling (Rao et al. 2000a, 2000b; Kangasjarvi et al. 2005). Depending on the severity of the stress, some O3 exposed plants exhibit typical symptoms of fumigation including enhanced leaf senescence, visible foliar injuries, reduced carbon assimilation, impaired stomatal conductance and reduced water-use efficiency (Martin et al. 2000; Joo et al. 2005). These symptoms helped to differentiate plant genotypes into ozone-tolerant and ozone-sensitive ones (Overmyer et al. 2000; Vahisalu et al. 2008). This genetic approach provided important resources and insights for dissecting the genetic networks underlying ozone stress response and tolerance (Tamaoki et al. 2003a; Kangasjarvi et al. 2005; A Ludwikow, unpubl. data, 2008; Vahisalu et al. 2008).

Due to the complexity of the ozone stress signal, deciphering ozone stress tolerance mechanisms has remained a major challenge to plant biologists. To develop new approaches to study plant response to ozone, a huge effort has been made to understand the molecular mechanisms that plants have evolved to survive these adverse conditions. The rapid progress of plant genomics provided new knowledge about how plants respond and adapt to O3 stress (Tamaoki et al. 2003b; Ahlfors et al. 2004; Gadjev et al. 2006; Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008). Significantly, available microarray experiments indicated genes with diverse functions that may act in regulation of ozone stress response and tolerance (Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008). Therefore, in this review we summarize the recent progress in understanding the transcriptome profile of ozone. We focused our consideration on microarray analyses identifying the gene networks responsible for response and tolerance to elevated ozone concentration. Of particular interest was the expression of genes encoding the enzymes involved in the metabolic pathway and genes related to stress signaling. The other aspects of plant response to ozone seen from different perspectives are available in the most recent reviews by Baier et al. (2005), Foyer and Noctor (2005) and Kangasjarvi et al. (2005).

Transcriptomic Approaches for Understanding of the Ozone Stress Response

The considerable development in the transcriptomics of ozone gives an overview of the various analytical and biological approaches that facilitate the identification of gene expression profiles in response to ozone exposure. Mahalingam et al. (2003) used PCR-based suppression subtractive hybridization to identify Arabidopsis genes that are differentially expressed in response to ozone, pathogens and the signaling molecules, showing a total of 1 058 differentially expressed genes from eight stress cDNA libraries with more than two-thirds of the genes in the stress cDNA collection not being identified in previous studies as genes with stress-related signatures. With the increasing availability of gene expression microarray technology, DNA microarrays (oligonucleotide and cDNA micro- and macroarrays) have been extensively used to identify ozone-inducible genes in a few plant species, including Arabidopsis thaliana (Tamaoki et al. 2003a, 2003b; Ahlfors et al. 2004; Ludwikow et al. 2004; Miyazaki et al. 2004; Mahalingam et al. 2005; Overmyer et al. 2005; Li et al. 2006; Gadjev et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008), Thellungiella halophila (Li et al. 2006), aspen (Gupta et al. 2005) and pepper (Lee and Yun 2006). Briefly, the insights gained from these multiple studies provided a more functional explanation of the regulatory network in ozone stress transcriptional responses and their regulation. Importantly, the transcriptomics analysis of ozone shows how the broad applications of available bioinformatic tools in microarray analyses can be used to validate or unravel new information about gene pathways (Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008), in-species (Gadjev et al. 2006; Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008) and cross-species gene-expression comparison (Gupta et al. 2005), and for the characterization of unknown relationships between genes (Ludwikow et al. 2008) as well as for identification of the cellular functions responsible for different ozone-related phenotypes (Tosti et al. 2006; A Ludwikow, unpubl. data, 2008). As expected, microarrays are indeed a powerful tool for the discovery of pathways and regulatory mechanisms in ozone stress response. Despite the fact that some minor discrepancies of the results exist and these are mostly due to the aims of the given experiment, the sets of arrayed genes, stress treatment conditions (e.g. ozone dose), filtering used, data analysis methods, or more important possible distinct ozone responsive mechanisms that are operating (Vahala et al. 2003; Li et al. 2006).

The analysis of trembling aspen trees by the poplar 4608 element array used to study long term responses of the tree to the effects of elevated CO2 and O3 showed that among 185 genes that were upregulated or downregulated in at least one of the treatments, 95 genes (88 upregulated and seven downregulated) responded to the elevated O3, whereas 51 genes change their expression pattern in response to the combined CO2 and O3 treatment (Gupta et al. 2005). Several functional categories of genes were identified to have particular expression profiles in ozone stress conditions. Genes with higher expression were related to signaling and defense categories. On the contrary, lower expression was observed for photosynthesis- and energy-related genes. In pepper, the 5K custom cDNA microarray was used to monitor the ozone-regulated genes in two ozone-sensitive and tolerant pepper cultivars (Lee and Yun 2006). Sixty-seven percent of the identified genes were regulated differently in the ozone-sensitive and -tolerant cultivars. Notably, most gene transcripts were found to be upregulated in the ozone-sensitive cultivars.

Microarray studies of A. thaliana further reveal the complexity and functional diversity of ozone stress response. These studies used a broad range of available designs and microarray platforms, providing useful information for the further dissection of signaling pathways involved in the perception or integration of ozone stress response in plants. Mahalingam et al. (2005), monitoring gene expression profiles in the time course microarray experiment (3 h, 6 h, 9 h, 12 h), identified 200 significantly expressed genes grouped into three different gene expression profiles: early upregulated (81 genes), late upregulated (60 genes), and downregulated (59 genes). These expression patterns correlated with particular Gene Ontology (GO) categories, revealing new patterns in the temporal evolution of the genetic response to oxidative stress. In his later study, ozone-sensitive Wassilewskija (Ws-0) ecotype of A. thaliana was used to analyze oxidative signaling elicited by an acute ozone dose (Mahalingam et al. 2006). In total, 371 genes were found differentially expressed at five different time points (1 h, 4 h, 8 h, 12 h, and 24 h) with different induced/repressed gene ratios depending on the time point.

Furthermore, a cDNA macroarray was also used in the analysis of ozone response. By using this platform, a complex hormonal crosstalk interaction between ethylene, jasmonic acid and salicylic acid signaling pathways was revealed in the regulation of ozone stress response (Tamaoki et al. 2003a, 2003b). In addition, the expression of 127 defense-related genes was studied in the rcd1 mutant and Col-0 plants by a customized cDNA macroarray (Overmyer et al. 2005) to identify genes differentially regulated in the mutant compared with wild type (WT) upon exposure to 250 nL/L O3.

Five other microarray analyses using oligonucleotide microarrays have been carried out. These studies, as most mentioned above, were not aimed at the simple identification of ozone-regulated genes, but were designed to reveal coordinated response among members of the same multigene family, associated with expression in oxidative stress conditions (Ludwikow et al. 2004; Tosti et al. 2006); to assess the specificity of ROS signaling in nine different ROS-generated systems (Gadjev et al. 2006); or were carried out to monitor the ecotype-specific expression profiles and implicate the involvement of multiple factors that contribute to the severity of ozone damage (Li et al. 2006). The effect of chronic ozone exposure on three A. thaliana ecotypes (Ws, Col-0 and Cvi-0) and T. halophila was analyzed by Li et al. (2006). This work confirmed the correlation between resistance capacity and accumulation of constitutive defense-related transcripts. However, more important is that the observed expression profiles in chronic ozone dose distinguish states and pathways associated with susceptibility (Ws), sensitivity (Col) and resistance to ozone (Cvi-0) as well as provide evidence of different stress-response kinetics and pathways activation, as opposed to acute ozone stress (Vahala et al. 2003; Kangasjarvi et al. 2005; Overmyer et al. 2005).

The most recent microarray experiment was designed to advance the understanding of interactions between abscisic acid (ABA) and ROS signaling pathways in O3 stress conditions (Ludwikow et al. 2008). This approach resulted in the identification of genes and candidate pathways involved in O3 stress response that are regulated by ABI1 protein phosphatase PP2C, known for its involvement in ABA signaling as its negative regulator (Saez et al. 2006; Yoshida et al. 2006). A comparison of the abi1td mutant strain with the wild type in ozone and drought shows the distinguishing effects of these two treatments on gene expression. The analysis of the abi1td-deregulated dataset emphasizes several subgroups with different gene expression patterns that correlated with various biological functions. Significantly, the comparison of the O3 and drought stress response in abi1td enabled the identification of new processes in O3-treated plants.

Functional Classification of Ozone-regulated Genes

Functional assignment differentially expressed genes, covering the broad range of annotation tools and strategies, become highly efficient in identifying the physiological and metabolic features of plant response to ozone (Miyazaki et al. 2004; Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008). A number of possible functions have been annotated to the differentially expressed genes that can be summarized into several major groups (Figure 1). Beyond the induction of highly expected genes involved in responses to hormone stimulus, general defense reactions, redox control, signaling and transcription, transport and the alternative splicing of the whole-genome expression profile encompass other response classes that are likely to be essential for plant survival in ozone stress. Among these predominant ones are genes that are associated with primary and secondary metabolism. Overall, these results provided new information on the adaptation of the metabolic networks to ozone stress conditions. Further functional analyses of these genes should provide a rich source of information on the mechanism of ozone stress response and tolerance.

Figure 1.

Model of biological processes involved in plant adaptation to ozone stress.
The graph shows the most significant biological processes identified in ozone transcriptome. The graph was generated from functional annotations provided in available microarray data. ABA, abscisic acid; AUX, auxine; BR, brassinosteroids; ET, ethylene; G, gibberellins; JA, jasmonic acid; MAPK, mitogen activated protein kinase.

Gene Expression Patterns for Metabolic Pathways

Metabolic regulations investigated from the viewpoint of gene expressions revealed new genes and pathways that control or adapt plant metabolisms to ozone stress. A general conclusion from these studies is that changes in metabolism are complex and involve multiple pathways. As illustrated in available microarray studies, cell metabolism adapts to ozone stress by the transcriptional activation of at least several genes in each pathway. The genes associated with sucrose biosynthesis, cell wall metabolism, glycolysis, pentose phosphate pathway, lipid metabolism, amino acid and protein metabolism, photorespiration, the shikimate pathway and its derivates, as well as genes coding for enzymes of the Tricarboxylic acid (TCA) cycle were affected significantly in response to ozone (Gupta et al. 2005; Mahalingam et al. 2005; Li et al. 2006; Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008). Several processes (suppression of protein synthesis; the repression of photosynthesis; the induction of sink metabolism and protein turnover) were already recognized as being a general response of plants to exogenous stressors (Li et al. 2006; Potters et al. 2007; Ludwikow et al. 2008). Others were found to be specific to ozone stress conditions (Ludwikow et al. 2008). For example, in comparison with drought stress treatment, genes related to phosphate and amino acid metabolism were significantly enriched in the O3 transcriptome of the ABI1 knockout. Among identified metabolic pathways, hormone synthesis (Kangasjarvi et al. 2005), metabolism of phenolic compounds (Saleem et al. 2001; Pasqualini et al. 2003; Samuel et al. 2005; Yaeno et al. 2006) and polyamines (Suorsa et al. 2002; Navakoudis et al. 2003; Kuznetsov et al. 2006; Groppa and Benavides 2008) have already been in particular interest of ozone studies. Recent microarray experiments have improved our understanding of their regulation in ozone stress conditions.

Hormone biosynthesis

Plant hormones ethylene (ET), jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) play an important role in the regulation of ozone stress response (Tuominen et al. 2004; Gomi et al. 2005; Yaeno et al. 2006; Ludwikow et al. 2008) (Figure 2). All play a major role in influencing plant growth and development, as well as plant responses to different stress factors (Balbi and Devoto 2008; Flors et al. 2008; Guo et al. 2008). As expected, transcriptiomic approaches identified new genes involved in plant hormone biosynthesis in ozone stress conditions.

Figure 2.

Regulation of hormone biosynthesis in ozone stress.
Ozone stress induces ethylene (ET), salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) biosynthesis. Both antagonistic and synergistic interactions among ET, JA, SA and ABA biosynthesis or signaling pathways were demonstrated. ABI1 PP2C regulates ozone-induced ABA and ethylene biosynthesis by the regulation of NCED3 gene expression and ACC synthase activity/stability. Interaction of ABI1 with components of mitogen activated protein kinase (MAPK) cascade that are involved in ET production are not excluded.

The activation of ET biosynthesis by the induction of the genes encoding 1-aminocyclopropane-1-carboxylate synthase (ACS6) is one of the fastest responses to O3 (Kangasjarvi et al. 2005). In Arabidopsis, the transcriptomic studies confirmed the rapid and transient activation of ACS6 in response to low and acute ozone doses (Ludwikow et al. 2008). However, the ozone-induced ethylene biosynthesis was also found to be modulated by other members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase family, including AtACS2 and AtACS10 (A Ludwikow, unpubl. data, 2008). The involvement of ACS2 has been postulated previously for its role played in the negative feedback regulation of ethylene synthesis (Wang et al. 2002). Reduced accumulation of the ACS2 transcript was observed in the ozone-treated ABI1 knockout plants (Ludwikow et al. 2008). However, these observations had a minor effect on ethylene evolution, since the abi1td mutant plants exhibited higher ethylene production compared with WT. Therefore, it is not excluded that the decrease of the ACS2 transcript level in the ABI1 knockout reflects a secondary effect, which compensates for higher ACS activity and the higher stability of ACS in the phosphorylated state, which was previously found to increase the rate of ethylene production (Liu and Zhang 2004). The steady state level of the ACS10 transcript has been found to be either upregulated or downregulated by biotic and abiotic stress treatments as well as plant hormones (Arabidopsis eFP Browser). In addition, ACS10 has been implemented in the regulation of flowering in response to light (Samach et al. 2000). The expression pattern of other ethylene biosynthetic genes such as 1-aminocylopropane-1-carboxylic acid oxidases (EFE, ACO2) and S-adenosylmethionine synthetase (SAM1) has been established in ozone stress conditions (Tosti et al. 2006; Ludwikow et al. 2008; A Ludwikow, unpubl. data, 2008). However, since it is known that the regulation of ACC synthases is essential for ozone-induced ethylene biosynthesis (Liu and Zhang 2004; Kangasjarvi et al. 2005; Ludwikow et al. 2008) the activity of other genes has not been an intensively studied.

Jasmonic acid is ubiquitous in plants and is normally connected with wound responses, developmental processes and senescence (Shan et al. 2007; Balbi and Devoto 2008). Although the biosynthetic pathway of JA is rather clear (Creelman and Rao 2002), a lot of effort is focused on resolving the crosstalk regulation of JA biosynthesis in ozone (Rao et al. 2000a, 2000b; Tuominen et al. 2004). O3 triggers and modulates JA biosynthesis via the modulation of expression for most known genes involved in the process, including lipoxygenases (LOX1–3), allene oxide synthase (AOS) and 12-oxo-phytodienoate reductases (OPR) (Rao et al. 2000; Tosti et al. 2006). Other genes that emerged form available ozone studies are S-adenosyl-L-methionine:  jasmonic acid carboxyl methyltransferase (JMT), OPC-8:0 CoA ligase1 (OPCL1), 3-ketoacyl-CoA thiolase (PED1), stearoyl-ACP desaturase and allene oxide cyclases (AOC1, AOC3) (A Ludwikow, unpubl. data, 2008).

Salicylic acid is a common plant phenolic compound and influences numerous physiological and biochemical processes in plants (Guo et al. 2008). The biosynthesis and metabolic pathway of SA is not completely understood. Although SA biosynthesis is mediated by isochorismate synthase (ICS1) and the phenylalanine ammonia lyase (PAL) pathway in ozone, SA biosynthesis seems to be synthesized mainly by the phenylalanine pathway (Ogawa et al. 2007). Other gene products such as EDS5, PAD4, SID2, and EDS4 have also been implicated in the SA biosynthetic process in plant disease resistance (Feys and Parker 2000; Ogawa et al. 2007). The accumulation of free SA is also associated with the formation of SA conjugates such as the SA glucoside and the Glc ester, as well as methyl salicylate (MeSA), a volatile SA ester (Lee et al. 1995). In ozone stress, the emission of MeSA was already studied in ozone-treated Scots pine (Pinus sylvestris L.) and tobacco plants (Nicotiana tabacum L. cv. Bel B and Bel W3) (Heiden et al. 1999). MeSA emission from Arabidopsis leaves was detected following the application of particular stresses to the plant (Van Poecke et al. 2001; Chen et al. 2003). Microarray analysis revealed the modulation of the BSMT1 gene expression coding for enzymes involved in the biosynthesis of MeSA. Nevertheless, little is still known about the biosynthesis and specific biological function of MeSA in plants. It has been postulated that MeSA may serve as a signal that activates resistance in nearby plants.

So far, there has been only one report that ABA levels increase upon ozone exposure (Bianco and Dalstein 1999). Importantly, recent transcriptomic studies identified a few genes involved in the regulation of ABA biosynthesis in ozone stress treatment, and in respect to this observation also uncovered a signaling pathway that modulates the crosstalk interactions between ABA and ET signaling and biosynthesis pathways (Ludwikow et al. 2008). It has been established that late ozone stress leads to changes in the expression of several ABA biosynthetic genes; O3 upregulates 9-cis-epoxycarotenoid dioxygenase NCED3 and downregulates ABA1, ABA2, ABA4, NCED1 gene expression. The NCED3 gene expression was crucial for the elevation of ABA synthesis in WT Col-0. The reduced accumulation of the NCED3 transcript resulted in lower basal levels of ABA in the ABI1 knockout and abolished the ABA biosynthesis in the ozone-treated ABI1 knockout plants. These results suggested that ABI1 is not only essential for O3 stress-induced ABA elevation but is also required for the maintenance of a basal level of ABA synthesis in A. thaliana Col-0 plants.

Although the ABA levels in the ozone-treated abi1td plants are decreased, the ethylene production, on the contrary, is significantly higher than in WT and comparable to the ethylene overproduction rate in the ein2 mutant (Ludwikow et al. 2008). These findings show that the ABI1 phosphatase antagonistically balances the biosynthesis of ABA and ET, and affects the ABA and glucose signaling pathway but not the ET signal transduction (the abi1td mutant did not exhibit overexpression of ethylene-regulated genes). Nevertheless, the mechanism has not been resolved yet. However, this showed that the ABI1 control of ethylene production was independent of ethylene perception, suggesting that there are at least two pathways controlling ethylene synthesis in O3-treated plants. Notably, other genes involved plant hormone biosynthesis or methylation were also identified as ABI1 downstream targets (Ludwikow et al. 2008). Overall, all of these findings suggest that ABA signaling and its interaction with ROS signaling is an important mechanism regulating ozone stress response.

The phenylpropanoid metabolism

Reports of plant molecular responses to ozone have pinpointed modulation in the activity of several genes of the phenylpropanoid pathway in different plant species (Saleem et al. 2001; Dixon et al. 2002; Pasqualini et al. 2003; Samuel et al. 2005; Yaeno et al. 2006; Zabala et al. 2006). This metabolic sequence channel produces a number of defense metabolites, involved as defense elements against biotic and abiotic stresses (Dixon et al. 2002; Samuel et al. 2005; Zabala et al. 2006). A metabolically important position, linking the phenylpropanoid secondary pathway to the primary metabolism (the shikimate pathway), the regulation of the overall flux into phenylpropanoid metabolism has been suggested to be modulated by the phenylalanine ammonia lyase (PAL), which is also required for SA synthesis (Ogawa et al. 2007). Several other genes leading to the synthesis of lignin (O-methyltransferase, coumarate CoA-ligase and cynamyl alcohol dehydrogenase), flavonoids (flavonol synthase, flavanone-3-hydrolase, flavonol 3-O-glycosyltransferase) or camalexin (PAD3) have been also shown to be modulated in ozone stress (Ludwikow et al. 2004; Tosti et al. 2006; Li et al. 2006). However, we lack a detailed analysis of phenylpropanoid compounds; available studies indicated both an accumulation and decrease of these metabolites in response to ozone.


Polyamines (PAs), spermidine (Spd), spermine (Spm), and their precursor putrescine (Put), have been described as endogenous plant growth regulators or intracellular messengers mediating physiological responses (Fig 4). As radical scavengers and protectants against environmental stresses, including ozone damage, polyamines (spermidine and putrescine) play pivotal roles in plant defense and tend to increase in response to any stress (Langebartels et al. 1991; Alcázar et al. 2006). Ozone modulates the transcription of at least four genes involved in polyamine synthesis. In tobacco Bel W3 plants, the accumulation of putrescine correlated with the induction of arginine decarboxylase (ADC) and ornithine decarboxylase activity (van Buuren et al. 2002). In A. thaliana, a drastic downregulation of two other key enzymes involved in spermidine biosynthesis: S-adenosylmethionine decarboxylase (SAMDC) and spermidine synthase (SPDS) was reported (Tosti et al. 2006).

Figure 4.

The polyamine biosynthesis pathway.
The polyamine biosynthetic pathway consists of four highly-regulated or constitutive enzymes: arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC) and spermidine synthase (SPDS). Expression levels of all of these enzymes are modulated by ozone treatment. The relationship between polyamines, γ-aminobutryic acid GABA and ethylene biosynthesis from S-adenosyl methionine (SAM) are also indicated. For further explanation see text.

In the past few years, attention has been focused on the role of PAs as regulators of stress signaling pathways. A crosstalk interaction between polyamine and ET biosynthesis has been established as a possible mechanism for preventing ozone damage (Wang et al. 2002). The accumulation of conjugated putrescine as a part of the ozone-induced programmed cell death response in ozone-sensitive tobacco Bel W3 plants was demonstrated (van Buuren et al. 2002). Additional evidence supporting polyamines as a metabolic signal building-up tolerance to stress conditions was also reported. Kasukabe et al. (2004) showed that the overexpression of spermidine synthase enhanced tolerance to various stresses including chilling, freezing, salinity, hyperosmosis, drought, and paraquat toxicity because of the increase in spermidine synthase activity and accumulation of spermidine content. Additionally, microarray analysis revealed a group of genes that were more abundantly transcribed in the SPDS overexpressor than in the wild type under chilling stress, suggesting an important role for spermidine as a regulator in stress signaling pathways.

Signal Transduction Pathways

The existence of complex signal transduction pathways in the response of plants to ozone stress is strong evidence for the complex hormonal control of O3 responses. Most of the ozone-responsive genes are induced by the plant hormones ET, JA, SA and ABA, and the balance between them determine the degree of plant ozone sensitivity and lesion formation (Baier et al. 2005; Kangasjarvi et al. 2005).

A bioinformatics-based functional analysis showed that the hormone-related groups of genes that undergo transcriptional regulation in ozone, represent different gene categories, including genes of unknown function as well as genes previously recognized as positive or negative regulators of hormone signaling, their effectors and marker genes associated with the activation of specific branches in a hormone signaling pathway (Tamaoki et al. 2003a, 2003b; Li et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008). In general, the analysis of these genes revealed subsets of ET-, JA-, SA- and ABA-regulated genes, respectively (Tamaoki et al. 2003a, 2003b; Li et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008) as well as groups of genes simultaneously regulated by more than one hormone (ET, JA and SA) (Tamaoki et al. 2003a, 2003b). Significantly, these recent transcriptomic studies have advanced our understanding of plant hormone interaction by providing information about the involvement of other hormones such as auxine (AUX), brassinosteroids (BRs) and gibberellins (G) (Mahalingam et al. 2006; Tosti et al. 2006; Ludwikow et al. 2008) in the potential regulation of ozone stress response, giving further support for extensive hormonal crosstalk in ozone stress response. Importantly, the studies were aimed not only at the identification of hormone-related genes but through a comparison of ozone-sensitive and -tolerant genotypes revealed implications to the basis of mechanisms determining ozone tolerance. Tamaoki et al. (2003a, 2003b) based on his transcriptional analysis of A. thaliana Columbia Col-0 and the jar1–1, ein2–1 and the npr1–1 mutant showed that a large number of ET and JA-regulated cell rescue/defense genes are suppressed by the SA pathway. Li et al. (2006) revealed that the expression of many of the ET-, JA-, SA-regulated genes are specific to a particular A. thaliana ecotype and seems to reflect the differences in sensitivity to chronic ozone exposure. To support this notion, Tosti et al. (2006), discussing the basis of the ozone-tolerant phenotype of Col-0 suggested that cell death promotive SA signal might not be fully activated in an ozone-exposed Col-0 plant, therefore some branches of SA signaling do not result in cell death.

The emerging picture of complex hormone interaction in ozone stress response showed that ABA also plays an important role as signal mediator for the activation of multiple downstream events that are important for ozone tolerance (Joo et al. 2005; Kangasjarvi et al. 2005; Vahisalu et al. 2008). ABA is essential for various stress responses, predominantly those associated with stomatal movement, metabolic changes and gene expression. Recent evidence suggests that in O3 stress, ABA functions as a crucial component of plant response determining O3-lesion initiation (Ahlfors et al. 2004; Kangasjarvi et al. 2005). The function of ABA has been found to be related to the regulation of O3 flux through stomatal pores. Notably, several regulators of ABA signaling in guard cells have been already implemented in the regulation of O3-stress response and tolerance, including the α or β subunit of the heterotrimeric G-protein as well as the putative G-protein coupled receptor and a plant guard cell S-type anion channel SLAC1 (Wang et al. 2002; Joo et al. 2005; Vahisalu et al. 2008). Furthermore, ozone stress activates the expression of numerous ABA-related genes (Ludwikow et al. 2008), which belong to distinct biological categories and in comparison to drought seems to be rather specific.

It has been postulated that ABA may also be an important component of O3-induced cell death (Kangasjarvi et al. 2005; Ludwikow et al. 2008). The protective role of ABA in programmed cell death (PCD) during endosperm development was reported (Fath et al. 2002; Steffens and Sauter 2005) as well as the interactions between the ABA, ET and JA signaling pathways, including the restriction of ethylene synthesis by ABA (Ghassemian et al. 2000; Sharp and LeNoble 2002; Rosado et al. 2006; Adie et al. 2007). To support these notions, Ahlfors et al. (2004) reported that the control of ET biosynthesis by RCD1, which was also affected in glucose, JA and ET responses, may involve ABA. In the sugar response, the primary effect of the rcd1-1 mutation seems to be more likely in ABA than ET responses. In addition, the increased ET synthesis in rcd1 might downregulate JA responses. Ludwikow et al. (2008) demonstrated that ABI1 protein phosphatase acts to inhibit ethylene production and stimulate ABA synthesis. In addition, the disruption of ABI1 resulted not only in the downregulation of JA-related genes but also in glucose hypersensitivity. We speculate that by similarity to rcd1, the downregulation of JA-related genes in abi1td might be also a result of ethylene overproduction. Together, these results indicate a complex hormonal interplay between ABA, ET and JA signaling pathways, in regulation of ozone response and tolerance. Moreover, the interplay between ABA, ET and JA signaling pathways seems to be guided primarily by ABA rather than JA-ET signaling.

Alternative Splicing in Ozone Stress Response

Although many expression profiling microarray platforms and designs have evolved over the past few years (which have led to increased scientific understanding of the plant genome) we still lack microarrays that directly monitor individual alternative splice events in plants. Given the large contribution of splice variants to the plant transcriptome, their characterization would provide an insight into signaling pathways involved in the regulation of stress response and even stress tolerance. An attempt to understand the diversity of plant transcriptome in ozone stress has been recently made (Ludwikow et al. 2008), and resulted in the identification of a group of genes that show variant splicing in the ABI1 knockout. In this mutant, which is obviously characterized by an impaired phosphorylation/dephosphorylation ratio, the frequency of occurrence of the alternative donor site AS event (AltD) was underrepresented compared with the A. thaliana genome, suggesting that phosphorylation/dephosphorylation events might be meaningful for the recognition of an alternative splice site. Notably, the results presented in the study provided a platform for future research, giving significant information about the functional categories in the transcriptome that are enriched in genes affected by alternative splicing during ozone stress.

Cis-elements and Binding Factors Involved in Ozone Stress Response

Transcriptome data have given us an opportunity to explore the basic mechanisms for controlling cell signaling in the initiation of stress inducible gene expression. An investigation into the transcriptomic regulation of stress-responsive genes involves the promoter analysis of the differentially expressed genes to identify common regulatory elements. In ozone stress, several cis-acting regulatory elements and corresponding binding proteins have already been implicated in the transcriptional network. These include stress-, light-, hormone (salicylic acid-, ethylene-, ABA-, auxin)-response elements and cis-motifs regulating plant growth and development (Tosti et al. 2006; Mahalingam et al. 2006; Ludwikow et al. 2008). However, we still lack valuable information or direct evidence for their involvement in intracellular defense against ozone stress.

The AP2/EREBP family of transcription factors, the WRKY proteins and the NPR1-interacting TGAC Motif-Binding (TGA) transcription factors regulated by SA make up the group of trans-factors expressed in response to ozone. Among these, the involvement of the WRKY TF in ozone response is predominant and the best documented (Tosti et al. 2006). The WRKY proteins comprise a family of zinc finger-type transcription factors and like Ethylene Response Factors (ERFs) or TGA TF are involved in the regulation of gene expression during pathogen defense, wounding and senescence (Miao et al. 2004; Journot-Catalino et al. 2006). Many of the WRKY genes were also induced in response to salicylic acid treatment (Dong et al. 2003). The 60 amino acids WRKY domains with the WRKYGQK motif at its N-terminal part were shown to interact with the cis-acting W-box motif (TTGACY) (Zhang and Wang 2005). Expression profiling revealed that the expression of at least 16 of 70 known WRKY factors is strongly deregulated during ozone stress. Fifteen members of the WRKY family were found to be upregulated (WRKY6, WRKY15, WRKY18, WRKY22, WRKY25, WRKY26, WRKY30, WRKY33, WRKY39, WRKY40, WRKY45, WRKY46, WRKY53, WRKY55, WRKY75) and one was downregulated (WRKY60) in response to ozone (Tosti et al. 2006). Among those identified the WRKY6 protein seems to be relevant to senescence and pathogen defense. The transcriptional activation of SIRK was found to be dependent on WRKY6 function (Robatzek and Somssich 2002). Overexpression of the pathogen-induced WRKY18 gene leads to an amplification of developmentally regulated defense responses (Chen and Chen 2002). WRKY22 is an important downstream component of a mitogen activated protein kinase (MAPK) signaling cascade involved in the response to bacterial and fungal pathogens (Asai et al. 2002). WRKY25 and WRKY33 are also coupled to MPK4-regulated defense activation via the MPK4 substrate MKS1 (Andreasson et al. 2005). The WRKY18, WRKY40, and WRKY60 transcription factors have partially redundant roles in plant responses to the two distinct types of pathogens (Xu et al. 2006). The WRKY6, WRKY22, WRKY30, WRKY33 and WRKY46 genes are induced by methyl viologen treatment (Scarpeci et al. 2008). In addition WRKY30, WRKY46 and WRKY53 TF are significantly induced by OPDA (Taki et al. 2005). WRKY53 acts in a complex transcription factor signaling network regulating senescence-specific gene expression (Miao et al. 2004). The WRKY75 protein seems to be involved in regulating a nutrient starvation response and root architecture (Devaiah et al. 2007).

The ERF family, containing one AP2/EREBP domain, is one of the largest groups of transcriptional factors. Their conserved DNA binding motif is GCC-box (AGCCGCC) (Riechmann et al. 2000). Ozone induces the expression of ERF1, ERF2, ERF4, ERF5 and ERF11 (A Ludwikow, unpubl. data, 2008). In addition, Tosti et al. (2006) shows that ERF-responsive elements are significantly enriched in the promoters of ozone-responsive genes. ERFs were identified and implicated in many diverse functions in cellular processes including biotic (Berrocal-Lobo et al. 2002) and abiotic stimuli (Tosti et al. 2006), development (Chuck et al. 2002), regulation of metabolism (Zhang et al. 2005) and hormone responses. In the last case, ERFs were found to act downstream of ethylene, jasmonate, and ABA signaling pathways (McGrath et al. 2005; Yang et al. 2005).

Other binding sites were identified in the ozone-induced transcriptional network as well. The ABRE motif and G-box elements were previously implicated in ABA-, light-, glucose- and redox-regulated gene expression as well as cold salinity and drought stress response (Uno et al. 2000; Baier and Dietz 2005; Kim et al. 2004). DRE/CRT are cis-acting elements functioning in ABA-independent gene expression in cold salinity and drought stress response (Narusaka et al. 2003). Both the ABRE motif and DRE/CRT cis-acting elements were found to be present in ozone regulated genes (Tosti et al. 2006; Ludwikow et al. 2008). In addition, the G-box (CACGTG) and H-box (CCTACC) cis-elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal, SA as early responses to pathogen attack (Droge-Laser et al. 1997). All of these suggested an extensive crosstalk between ozone and other biotic and abiotic stress-signaling pathways. TGGGCY SITEIIATCYTC was overrepresented in the promoters of nuclear genes encoding components of oxidative phosphorylation (Welchen and Gonzalez 2006). The ANAERO1 consensus sequence AAACAAA (Mohanty et al. 2005), AGL1AT, L1-box, DOF domain proteins, A/T rich CArG motif were the binding site of the MADS-domain protein APETALA3 (Tosti et al. 2006); metal responsive element MRE1 (TGCRCNC), Light-regulated HDZIP2 (TAATMATTA) and T-box motif (Mahalingam et al. 2006). The heat-shock consensus element HSE (CTNGAANNTTCNAG) is found in multiple copies upstream of all heat-shock genes (Bharti et al. 2000).

Concluding Remarks

Overall, the results from the ozone transcriptomic studies exemplify the benefits that can be harvested from the application of integrative and systematic analytical approaches to study ozone stress response. Although substantial progress has been made on uncovering genes regulated by ozone, much work still remains to be done to completely characterize the association between gene-expression patterns and the ozone-induced phenotype. The results clearly show that there is an impressive range of mechanisms available in plants to counteract ozone stress. Plant ozone defense responses clearly depend on the interplay between many complex signaling pathways and metabolite signals. The picture arises of a signaling network where ET, ABA, JA and SA play a central role in the process. However, there are indications from microarray studies that defense responses might be turned on by other hormones. Furthermore, alternative splicing opens up the way to a highly complex picture of ozone-induced proteosome with many signals becoming an integrated part of ozone stress response. Fortunately, from all of the information available, several studies found evidence of an understanding of the transcriptional regulation of metabolite pathways. A continuation of their work will be pivotal for finding out whether and how these metabolites affect ozone response and which of them may serve as molecular signals switching pathways and interacting with each other. The approach of a biological system modeling in ozone stress is coming closer and closer.

(Handling editor: Qi Xie)