Common Links of Molecular Biology with Biochemistry and Physiology in Plants Under Ozone and Pathogen Attack
This chapter focuses on the transcriptional events of stress responses upon ozone and pathogen attack. The ozone effects of herbaceous and woody plants are compared at the promoter, transcript, protein and metabolite levels. The transcription factors involved in plant abiotic and/or biotic stress responses are shown. These different approaches will be discussed at the physiological level. In addition to microarray analyses, an integration of ozone effects on the shikimate pathway and ethylene biosynthesis is given. The transcriptional differences between the sun and shade leaves of European beech, the ontogenetic effects comparing juvenile and mature trees, and gene expression studies covering two vegetation periods are discussed.
KeywordsSuppression Subtractive Hybridisation Ethylene Biosynthesis European Beech Ozone Exposure Ozone Treatment
Ozone is a ubiquitous phytotoxic air pollutant that severely affects vegetation, including that of forest ecosystems. During the last decade, the effects of ozone and the mechanistic responses of plants at the level of gene expression have become clearer (Kangasjärvi et al. 1994; Langebartels et al. 1997, 2002; Dizengremel 2001 Mahalingam et al. 2003; Jaspers et al. 2005; Ludwikow and Sadowski 2008; Matyssek et al. 2008). Moreover, it has been shown that acute ozone exposure results in a gene expression pattern similar to pathogen attack, and the appearance of ozone-induced cell lesions resembles the hypersensitive response of plants, sharing many of its physiological and molecular features (Sandermann 1996; Pell et al. 1997; Rao et al. 2000; Jaspers et al. 2005; Heath 2008). Ozone exposure and biotic stress seem to have several commonalities. Therefore, ozone has been recognised as an abiotic elicitor of plant defence reactions (Sandermann et al. 1998), and a crosstalk between abiotic and biotic stress responses is evident (Eckey-Kaltenbach et al. 1994; Fujita et al. 2006). Furthermore, the transcriptional regulation of receptor-like protein kinases has also shown strong response similarities between ozone and pathogen stress (Wrzaczek et al. 2010).
Acute ozone exposure (200–300 nl l−1 for several hours) results in an up- or down-regulation of well-studied ozone-responsive genes (Matyssek et al. 2008). Microarray analyses have revealed that these transcripts could be categorised into functional groups belonging to different pathways of metabolism, such as primary metabolism, energy, cell growth and division, transcription, protein synthesis and destination, transport, cell structure, signal transduction, disease and defence, secondary metabolism, and non-categorised ones for Arabidopsis thaliana (Mahalingam et al. 2003; Tamaoki et al. 2003; Ludwikow et al. 2004; Tosti et al. 2006) and Medicago truncatula (Puckette et al. 2008, 2009). In addition, acute ozone exposure of ozone-sensitive Arabidopsis mutants showed that the response of plants towards ozone is under complex genetic regulation (Overmyer et al. 2008). Comparing acute and chronic (up to 100 nl l−1 for several weeks) ozone exposure of soybean plants it has been shown at the physiological level that changes in the photosynthetic capacity were very similar; however, a different pattern of ozone damage at the leaf level was evident (Chen et al. 2009). Experiments of chronic ozone exposure of up to twice the ambient level over several days, weeks or even years with herbaceous plants and forest trees have been performed under both controlled-chamber conditions (Langebartels et al. 1997; Miyazaki et al. 2004; D’Haese et al. 2006; Chen et al. 2009; Bohler et al. 2010) and open-air conditions, for example, the Aspen Free-Air Carbon Dioxide Enrichment (FACE) Experiment (http://aspenface.mtu.edu/), Kranzberg Ozone Fumigation Experiment (KROFEX; http://www.sfb607.de/) as part of the CASIROZ study (Matyssek et al. 2007; http://www.casiroz.de), the Kuopio open-field exposure system (Wulff et al. 1992; Karnosky et al. 2007), a lysimeter study (Winkler et al. 2009; http://www.helmholtz-muenchen.de/en/lysimeter/home/index.html) or the Soybean Free Air Concentration Enrichment (SoyFACE; http://soyface.illinois.edu/index.htm) experiment. In these free-air field experiments, some genes were similarly up- or down-regulated by chronic ozone fumigation, as compared to acute ozone exposure in the following analysed plants: A. thaliana (Miyazaki et al. 2004; Li et al. 2006), Thellungiella halophila (Li et al. 2006), trembling aspen (Gupta et al. 2005), European beech (Olbrich et al. 2009), and paper birch (Kontunen-Soppela et al. 2010b). However, differences in transcripts have also been observed that might reflect additional stress factors under field conditions (Miyazaki et al. 2004; Olbrich et al. 2010a).
At the transcriptional level, powerful techniques, such as microarray analysis, quantitative real-time RT-PCR, and, more recently, high-throughput sequencing methods, have been employed that can provide significant information about the transcribed genes that are involved in abiotic/biotic stress responses and the adaptation of plants to changing environments. However, the results have to be considered carefully, as transcript and protein levels are not necessarily and/or clearly correlated (Gygi et al. 1999). In addition, low-abundance mRNAs, often important in regulation processes, are not easy to detect and to quantify, in contrast to transcripts that are abundant. The analysis of proteins and of the proteome in response to different stresses is now possible through sophisticated techniques, such as 2D-DIGE, MALDI-MS/MS, LC-MS/MS or iTRAQ, which can provide information about the proteins encoded by the genes involved in these stress responses (Renaut et al. 2006; Jorrín-Novo et al. 2009). Plant metabolites are the end products of gene and protein expression and can reflect, at least in part, the final step of the plant’s response to environmental stress (Tretheway 2001). Therefore, integrated “omic” analyses are important to understand plant responses towards abiotic/biotic stress and to identify the regulatory networks in plant stress responses (Urano et al. 2010). The integration of transcript, protein and metabolite data will reflect the response of the plant to environmental stress at a profound and comprehensive level (Oksman-Caldentey and Saito 2005; Bohnert et al. 2006; Trauger et al. 2008).
2.2 Transcription Analyses upon Abiotic and/or Biotic Stress in Non-woody and Woody Plant Species
2.2.1 Transcription Analysis in Non-woody Plant Species upon Ozone Treatment
Acute ozone-induced transcriptional changes were first observed in an ozone-sensitive tobacco cultivar by analysing pathogenesis-related (PR) protein transcripts (Ernst et al. 1992; Schraudner et al. 1992). Thereafter, many single transcriptional studies have been conducted, reflecting antioxidant enzymes, ethylene biosynthesis, phenylpropanoid metabolism and diverse metabolic pathways, which are known to be affected by biotic stress (Kangasjärvi et al. 1994; Langebartels et al. 2002; Heath 2008). New technologies soon emerged, such as cDNA microarray, which have allowed the expression analyses of thousands of genes; indeed, in the model organism, A. thaliana, the whole transcriptome has been analysed. It was found that hundreds of genes were up- or down-regulated and could be grouped into functional categories of whole-plant metabolism (Ludwikow et al. 2004; D’Haese et al. 2006; Li et al. 2006; Tosti et al. 2006). Gene expression profiles of ozone-exposed A. thaliana revealed that the three hormones, ethylene, jasmonic acid and salicylic acid, interact in the regulation of ozone-affected genes (Tamaoki et al. 2003). This interaction, as a result of an oxidative burst, has many characteristics in common with the pathogen-caused hypersensitive response, which results in an altered gene expression for genes such as PR proteins, following both ozone and biotic stresses (Overmyer et al. 2003; Jaspers et al. 2005). The hierarchical clustering of ozone stress-regulated genes in pepper plants that were treated either with ozone or Xanthomonas axonopodis sp. glycines showed an extensive overlap of commonly affected genes (Lee and Yun 2006). In soybean, it was found that mainly defence-related genes, hormone signalling-related genes and genes encoding transcription factors were commonly regulated by elevated ozone and mosaic virus infection, and the highest gene expression changes were observed when both stressors were applied at the same time (Bilgin et al. 2008).
It is known that WRKY (conserved peptide sequence) transcription factors are involved in various plant processes that cope with abiotic and biotic stress and that there is a network of WRKY transcription factors in defence signalling (Eulgem and Somssich 2007; Pandey and Somssich 2009). For binding the WRKY transcription factor, the cognate cis-acting W box is essential (Eulgem et al. 2000). The W box motif (TGAC) and a W box-like motif were significantly over-represented in an ozone-induced cDNA library of A. thaliana (Mahalingam et al. 2003). Similarly, the promoter of the class I β-1,3-glucanase gene, which contains a GCC box, is induced upon ozone and pathogen stress (Leubner-Metzger and Meins 1999; Grimmig et al. 2003; Ernst and Aarts 2004). Several transcription factors, that are up-regulated by ozone, including WRKY, MYB (myeloblastosis) and the zinc finger protein family, have been described in herbaceous plants (D’Haese et al. 2006; Li et al. 2006; Tosti et al. 2006; Cho et al. 2008), with an over-representation of WRKY transcription factors, indicating again the remarkable similarities in gene expression induced by ozone and pathogen stress.
2.2.2 Transcription Analysis in Woody Plant Species upon Ozone Treatment
The specific conditions of microarray analyses require careful scrutiny, incorporating data pre-processing, data normalisation and statistical analysis to select differentially expressed genes, and Loess normalisation, principal component analysis and empirical Bayes methods are necessary in the analysis (Olbrich et al. 2009) (see Chap. 16 for the statistical methods, including support vector machines, for the classification of as yet unknown genes). Direct transcript correlations of woody and herbaceous plants have been performed for genes involved in ethylene biosynthesis (Betz et al. 2009a) and the shikimate pathway (Betz et al. 2009b). Janzik et al. (2005) have demonstrated that ozone had dramatic effects on the regulation of the shikimate pathway in tobacco. Furthermore, in European beech, an ozone-dependent reprogramming of the transcription of all shikimate pathway genes, as a prerequisite for secondary metabolism, has been demonstrated (Betz et al. 2009b).
Cis/trans elements are important for gene regulation, and an ozone-responsive W box has been described in the resveratrol synthase, Vst1, promoter of grape vine, which was also induced by Botrytis cinerea (Schubert et al. 1997; Grimmig et al. 2003; Ernst and Aarts 2004). Several ozone-induced WRKY transcription factors have been described in herbaceous plants and shrubs (Mahalingam et al. 2003; D’Haese et al. 2006; Heidenreich et al. 2006; Tosti et al. 2006; Paolacci et al. 2007; Cho et al. 2008). In trees, ozone-responsive WRKY transcription factors have also been described and were shown to be up-regulated in beech (Olbrich et al. 2005) and poplar (Rizzo et al. 2007) and down-regulated in paper birch (Kontunen-Soppela et al. 2010b).
2.2.3 Combining the Transcriptional Data of Beech, Apple and Potato
The establishment of suppression subtractive hybridisation (SSH) libraries from scab susceptible apple leaves strongly infected with Venturia inaequalis produced results that indicated that approximately 10 % of ESTs belonged to the category disease/defence (Zistler 2007), which is in good agreement with the data from ozone-treated European beech (Olbrich et al. 2005). In addition, genes coding for PR proteins and distinct heat shock proteins were also present and were in agreement with the results from European beech (Olbrich et al. 2005; Zistler 2007). Similarly, the sequence analyses of SSH libraries of susceptible and moderately resistant potato cultivars infected with Phytophthora infestans resulted in approximately 22 % and 9 %, respectively, of the ESTs belonging to the category of disease/defence (Ros et al. 2004). As was found for European beech and apple, genes encoding PR proteins were also identified as induced in potato (Olbrich et al. 2005, 2010b; Ros et al. 2004; Zistler 2007) (see Chap. 4 for defence genes induced by insects and Chaps. 5 and 10 for the positive interactions of microorganisms and mycorrhizal fungi). In conclusion, most genes belonging to the category disease/defence were up-regulated in the different plant species analysed upon ozone or pathogen treatment. However, it has to be kept in mind that the whole transcriptome was not analysed, as has been described for the model plant, A. thaliana. This deficiency might be supplemented by using next-generation sequencing that may provide a deeper insight into the whole transcriptome of plant genomes that, to date, have not been fully sequenced.
2.3 Correlations Between the Genome, Transcriptome, Proteome, Metabolome and Physiology upon Ozone Stress
Correlating the genome, transcriptome, proteome and metabolome data of plant responses to ozone stress is important to understand the changes in the biochemistry of the whole plant. Sandermann and Matyssek (2004) have provided a scaled-up model of the molecular and ecological processes in plants. In this excellent overview, the hierarchical levels and interactions between the molecular processes, cells and tissues, organs, whole-plant systems, and ecosystems and biochemical cycles are outlined, and the influence of environmental factors on each of these levels are discussed.
The integrated “omic” approaches are possible for the holistic analyses of model plants that have been fully sequenced, including A. thaliana, rice and poplar (Oksman-Caldentey and Saito 2005; Cho et al. 2008; Renaut et al. 2009). Although custom microarray chips are currently available, proteome/phosphoproteome and metabolome (non-targeted profiling) analyses are still highly sophisticated, difficult to conduct, and expensive. Only a few metabolic studies applied to ozone stress can be found in the literature; the species that have been investigated include white birch (Kontunen-Soppela et al. 2007; Ossipov et al. 2008) and rice (Cho et al. 2008). In white birch, ozone was found to cause increases in phenolic compounds and compounds related to cuticular wax layers, whereas compounds related to carbohydrate metabolism and chloroplast pigments were decreased; interestingly, the metabolite profiles showed differences between different birch genotypes (Kontunen-Soppela et al. 2007). Similarly, the differences within a single tree, between trees within an experimental field, or between different fields was shown to increase the phenotypic variation in silver birch metabolites, which may have led to difficulties in the interpretation of the results (Ossipov et al. 2008).
In contrast, more examples of proteomic data are available in the literature. Gel-based proteomics and immunoblotting techniques have identified bean and maize proteins that were also known to be induced by ozone at the transcriptional level (Torres et al. 2007). Similarly, MS/MS analyses have revealed known ozone-regulated proteins (Torres et al. 2007), and 2D-DIGE analyses have identified a large number of proteins involved in carbon metabolism, electron transport, detoxification, secondary metabolism, protein folding and disease/defence (Bohler et al. 2007, 2010). In soybean, the analyses of total soluble and chloroplast proteins have revealed that proteins involved in photosynthesis and carbon assimilation decreased, and proteins involved in carbon metabolism and antioxidant defence increased, after ozone stress (Ahsan et al. 2010). In rice and wheat, ozone was found to affect the levels of photosynthetic, antioxidative defence, disease/defence and stress-related proteins (Agrawal et al. 2002; Feng et al. 2008; Sarkar et al. 2010), all of which are factors of pathways that are known to be affected at the transcriptomic level. With regards to a holistic approach analysing transcriptomics, proteomics and metabolomics, only a single reference with respect to ozone stress exists in the literature thus far (Cho et al. 2008). It was found that genes categorised into cellular processing and signalling, information processing and storage and metabolism were mainly regulated; the identified proteins were found to be involved in cellular processing and signalling, photosynthesis and defence. On the basis of genes functionally categorised from transcriptome data, Cho et al. (2008) demonstrated an induction of the pentose phosphate pathway, ethylene and jasmonate biosynthesis, transcription of naringenin-related genes, and genes involved in glutamate and γ-aminobutyric acid biosynthesis. This systematic survey showed that ozone triggered a chain reaction of altered gene and protein expression and metabolite accumulation in rice (Cho et al. 2008).
An initial comparison of transcriptional and physiological data of tree growth following ozone treatment was obtained in the Aspen FACE experiment using trembling aspen (Karnosky et al. 2007). The treatment resulted in a pattern of gene expression that was found to fit well with the overall patterns of physiology and growth (Wustman et al. 2001; Karnosky et al. 2003; Gupta et al. 2005). Although protein and metabolite data were not included, these correlations were a breakthrough in the analysis of field-grown trees. Regarding the carbon metabolism of conifers, a review by Dizengremel (2001) showed reduced mRNA levels of the small and large subunits of ribulose 1,5-biphosphate carboxylase/oxygenase (Rubisco), a decrease in the protein levels of the large subunit, a decrease in the Rubisco relative activity and a decrease in the relative photosynthetic rate upon ozone treatment, indicating premature senescence. The long-term effects of elevated tropospheric ozone on silver birch have revealed an up-regulation of many senescence-associated genes, in correlation with an earlier abscission of leaves and a decreased chlorophyll content (Kontunen-Soppela et al. 2010a). However, in contrast to the decrease in the gene expression of photosynthesis-related genes and the amount and activity of Rubisco, net photosynthesis was not affected. Therefore, gene expression data do not necessarily reflect biochemical processes or plant physiology (Kontunen-Soppela et al. 2010a).
Stilbenes, a major constitutive of phenolic compounds in Scots pine, have been shown to accumulate in the needles upon abiotic and biotic stresses (Rosemann et al. 1991; Lieutier et al. 1996). In Scots pine seedlings, an ozone-induced increase of stilbene synthase (STS) transcripts, STS enzyme activity and stilbene content was demonstrated (Rosemann et al. 1991; Chiron et al. 2000a, b). Lignin, the second most abundant organic material in the biosphere after cellulose, is synthesised via cinnamylalcohol dehydrogenase (CAD). Ozone exposure of poplar leaves resulted in a rapid and strong increase of CAD mRNA levels and CAD enzyme activity, independent of the foliar development (Cabané et al. 2004). In addition, the lignin content was substantially increased under ozone exposure, which indicated a regulated correlation between transcripts, enzyme activities and stress-induced lignin content (Cabané et al. 2004).
At the Kranzberger Forst research facility, during the CASIROZ study with adult European beech, treatment with an ozone level that was twice the ambient level revealed an up-regulation of 9-cis-epoxycarotenoid dioxygenase, which encodes the key enzyme of abscisic acid (ABA) biosynthesis. This result was in accordance with the observed increase in the level of ABA (Jehnes et al. 2007; Matyssek et al. 2007). Consequently, decreased stomatal conductance and photosynthesis rates were observed in the leaves (Löw et al. 2007; Matyssek et al. 2007). However, an expected reduction of the annual stem growth on the basis of stem diameter measurements was not observed after 3 years of ozone exposure (Wipfler et al. 2005). In contrast, after 8 years of ozone fumigation at twice the ambient level, a drastic decrease in the volume growth was observed on the basis of tree-ring analysis and height-increment measurements (Pretzsch et al. 2010; Matyssek et al. 2010a, b; see Chap. 13 for discussions on allocation and allometry). Again, these results demonstrate that an extrapolation from genes to total-tree growth, even at an ecological level, needs a large number of data for a global perspective.
Ozone-induced ethylene biosynthesis has been shown to be the result of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACO activity (Kangasjärvi et al. 1997; Moeder et al. 2002). ACC synthesis is catalysed via ACS, and ACO, in turn oxidises, ACC to ethylene. In European beech, it was demonstrated that the emission of ethylene, the levels of its precursor, ACC, and the transcript levels of ACS2 and ACO1 showed a persistent increase and preceded cell death upon ozone fumigation (Nunn et al. 2005a). Similarly, it was shown that cell lesion formation in European beech leaves was preceded by persistent increases in ethylene emission, in the level of its free and conjugated malonylated precursor, ACC, and in transcript levels, specifically for ACS1, ACS2, ACO1 and ACO2 (Betz et al. 2009a). These results demonstrate a chain reaction of altered gene and metabolite expression and a change in leaf physiology/morphology.
Microarray analyses of leaves of European beech that were grown at an outdoor free-air model fumigation site showed down-regulated levels for transcripts belonging to photosynthetic-related, chloroplast cell structure and Calvin cycle genes for the years 2005 and 2006 (Olbrich et al. 2009). Subsequent proteome analyses also revealed reduced levels of proteins involved in photosynthesis and the Calvin cycle (Kerner et al. 2011). Although there was no direct overlap of distinctly regulated transcripts/proteins at a specific time point, both methods clearly indicated an overall down-regulation of primary metabolism upon ozone treatment. Similar results of transcriptomic and proteomic analyses were obtained with European beech roots after infection with P. plurivora, showing only two overlaps: phosphoglucomutase and cytochrome reductase (Schlink 2009; Valcu et al. 2009) (see Chaps. 3, 5 and 10 for more detail on processes for below-ground organs). In addition, the down-regulation of transcripts belonging to primary metabolism, photosynthesis and chloroplast cell structure, indicating an earlier senescence in ozone-treated trees at the end of the growing season, was correlated with phenological data on leaf senescence, as indicated by an earlier discoloration of the leaves (Pritsch et al. 2008).
2.4 Ontogenetic Effects and the Differences Between Sun and Shade Leaves
Comparing all of the transcriptional data from several years of field experiments using chronic ozone concentrations of up to twice the ambient level, it was found that the levels of changed transcripts were weak, compared to those found with acute, higher ozone concentrations (Miyazaki et al. 2004; Gupta et al. 2005; Li et al. 2006; Olbrich et al. 2009, 2010a; Kontunen-Soppela et al. 2010a, b). This indicates the importance of exposure to more realistic ozone concentrations in the field for ozone risk-assessment studies (Matyssek and Sandermann 2003; Karnosky et al. 2007; Matyssek et al. 2008). Because the sun crown of a tree is exposed to high photosynthetically active radiation and increased short-wave length UV radiation, as compared to the shade crown, differences in gene expression can be expected for light- and UV-regulated pathways (Zinser et al. 2007; Götz et al. 2010; see Chap. 8 for a discussion on solar radiation).
Transcriptional analyses for selected genes in beech leaves at the Kranzberger Forst research facility during 2003 and 2004 have indicated differences between the sun and shade leaves for genes involved in ethylene formation, stomatal closure and lignin biosynthesis (Jehnes et al. 2007; Matyssek et al. 2007; Matyssek et al. 2010b). However, significant differences in gene expression were only observed for ACS2, a gene involved in ethylene biosynthesis, and caffeic acid O-methyltransferase, which is involved in lignin biosynthesis (Jehnes et al. 2007). Using an in-lab-produced microarray (i.e. non-commercial) containing ozone-responsive ESTs, it was found that only 0.1–0.2 % of all of the ESTs appeared to be differentially expressed in sun and shade leaves (Olbrich et al. 2010a, b). Furthermore, a comparison of the ozone effects and leaf type on gene expression revealed a stronger influence of the leaf type than of the ozone treatment (Fig. 2.3; Olbrich et al. 2010b); however, no consistent differences at different time points within two consecutive years were detected (Matyssek et al. 2010b; Olbrich et al. 2010a). These results indicate that unpredictable factors, such as weather conditions, pathogens and nutrient supply, may strongly influence gene expression.
2.5 Concluding Remarks
Ozone acts as an abiotic elicitor in both herbaceous and woody plant species (Sandermann et al. 1998; Matyssek et al. 2005). This has been clearly demonstrated at the level of gene regulation and results in a complex interaction of cis/trans elements. W box motifs and WRKY transcription factors are known as crucial regulators of the changes in the transcriptome as a defence response in plants after pathogen attack, and they also act in the response of plants to ozone treatment. Therefore, it is not surprising that after both types of treatment, transcripts have shown a similar up-regulation for distinct biochemical pathways. Combining the transcriptional data of European beech, apple and potato has revealed that most of the genes belonging to disease/defence are up-regulated in these plant species upon ozone or pathogen stress.
Thus far, data of the responses of the entire transcriptome are very rare, and studies may be complemented by the utilisation of next-generation sequencing. A common response of the transcriptome, proteome and metabolome upon ozone treatment was shown as an up-regulation in the shikimate pathway and ethylene biosynthesis. Further insight into this complex regulation is expected by holistic, large-scale “omic” approaches and the application of bioinformatic tools. In addition, phosphoproteomics will also be a valuable tool to unravel plant regulatory mechanisms upon stress (van Bentem et al. 2006). For the first time, DNA-array analyses have shown the distinct differences between sun and shade leaves, as well as between the leaves of juvenile and mature trees grown under free-air ozone exposure. In addition, transcriptional differences between different years were observed, indicating that less predictable factors, given by variations in the environment, may influence gene expression. Moreover, the combined effects of different abiotic stresses under field conditions should be a focus of future research, as combined stress effects may show positive or negative interactions (Mittler 2006; Eastburn et al. 2011).
In recent years, it has been demonstrated that long, non-protein-coding RNAs and small RNAs are major regulators of gene expression (Jacquier 2009; Charon et al. 2010; Chitwood and Timmermans 2010). Small RNAs are important in biotic stress responses, mostly as mediators of repressive gene regulation through RNA silencing (Ruiz-Ferrer and Voinnet 2009). Stress-responsive microRNAs for cold, heat, dehydration and UV-B radiation have been described in Populus (Lu et al. 2008; Jia et al. 2009). Novel mechanical stress-responsive microRNAs in poplar, which are absent from Arabidopsis, have also been described (Lu et al. 2005). These results have provided new insights into the regulatory networks that modulate the interaction between the plant genome and environmental factors. Ozone effects may, therefore, also be further regulated by such microRNAs, and this will be a challenge for further research. Similarly, gene expression driven by stress factors often depends on histone post-translational modifications and DNA methylation, which accounts for epigenetic gene regulation (Chinnusamy and Zhu 2009; Williams 2011). An epigenetic variation in trees occurring in contrasting environments or affecting climatic adaptation has also been described (Johnson et al. 2009; Lira-Medeiros et al. 2010). Interestingly, memory and carryover effects in the action of ozone on conifers have been portrayed with delayed physiological effects appearing months after the initiation of the treatment, even at a near ambient level of ozone fumigation (Sasek et al. 1991; Langebartels et al. 1998; Oksanen 2003). However, the degree to which extent the memory effects and corresponding imprinting are inherited or acquired remains unknown, even though it has been described in various plants (Ries et al. 2000; Molinier et al. 2006; Lang-Mladek et al. 2010). The resolution of this question will be a major challenge for ongoing research projects.
We are grateful to our co-workers for their important contribution during the past years. We thank Ana Staninska and Gerhard Welzl for their help with statistical methods. Special thanks go to Pierre Dizengremel (Nancy-Université) and Elina Oksanen (University of Eastern Finland) for critically reading the manuscript and helpful comments. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 607) and, in part, by the European Community (Evoltree, 6th Framework Programme; COST Action E52).
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