The ozone-induced oxidative burst correlates with and precedes the HR-type injury in various plant species
In addition to triggering antioxidant defence responses, ROS are involved, particularly at higher concentrations, in the most drastic plant defence response, the suicide of pathogen-invaded cells (Lamb & Dixon 1997). Here we show that an environmental contaminant, the air pollutant ozone, triggers the accumulation of ROS in ozone-sensitive plant species. Our findings provide evidence that these plant-derived ROS are involved in the formation of HR-type lesions found in plants exposed in climate chambers as well as in the field.
As exemplified with the ozone biomonitor plant, tobacco Bel W3, the patterns of ozone-induced H2O2 accumulation, cell death and visible symptoms were identical with regard to tissue and developmental stage specificity (Fig. 1). Initially, tobacco symptoms were described as ‘weather flecks’, and they were found on the adaxial side of the leaf, in the vicinity of first- and second-order veins (Lucas 1975). The air pollutant ozone was identified as the causal agent for these symptoms in a classical paper by Heggestad & Middleton (1959). Figure 1 shows that the sites of H2O2 accumulation in the ozone-sensitive tobacco Bel W3 correlate with visible symptoms in all developmental stages. Ozone injury in tobacco specifically starts in the palisade parenchyma cells (Guderian 1985). Histochemical analysis of cross-sections of leaves revealed that the palisade cells were also the sites of H2O2 accumulation. Using the non-permeable dye hexafluorofluorescein covalently linked to bovine serum albumin, it could be demonstrated that the accumulation initially occurs extracellularly, that is, in the cell wall and the apoplastic fluid surrounding the palisade cells (Fig. 1). These results confirm the subcellular location of ROS production in ozone-exposed birch leaves in which ROS were initially also detected in the cell wall and on the plasma membrane (Pellinen et al. 1999).
A survey of various species revealed that H2O2 and O2–• accumulation, detected by DAB and NBT staining, respectively, appeared well before the HR-type cell death in ozone-sensitive plants. Sites of H2O2 accumulation correlated spatially with those of cell death in nine tomato cultivars, whereas O2–• sites correlated with lesions in M. sylvestris. All assays were carried out 2–3 h after the end of the exposure period which excludes ozone-derived ROS as the cause for DAB and NBT staining. In addition, pre-treatment of tomato, A. thaliana and M. sylvestris plants with DPI, a suicide inhibitor of flavin-containing oxidases such as the superoxide-producing NADPH oxidase, markedly reduced ROS accumulation and cell death in parallel (Figs 5 & 6). Similarly, DPI abolished endogenous ROS production in pathogen-challenged plants (Levine et al. 1994; Jabs et al. 1996). In addition, the protein kinase inhibitor K252a reduced H2O2 accumulation and cell death in tomato (Fig. 5) suggesting an involvement of protein kinases in the ozone-induced oxidative burst (see also Samuel, Miles & Ellis 2000). Taken together with previous results (Schraudner et al. 1998; Rao & Davis 1999; Overmyer et al. 2000), it appears that ozone triggers the endogenous production of ROS in sensitive plants, thus amplifying the direct effects of ozone in discrete tissues and stages of development. In line with these findings, the onset of cell death in several lesion mimic mutants is tissue- and developmental stage-specific (McDowell & Dangl 2000; Glazebrook 2001).
As summarized by Wojtaszek (1997), the oxidative burst in plant–pathogen interactions is often composed of the more stable H2O2, but also O2–• accumulation is found in certain systems. Accordingly, the type of the predominant ROS differed between the species investigated in this study even though they were exposed under similar climate and pollutant regimes, and were all tested with DAB and NBT staining under identical conditions. Whereas the tobacco and all tomato cultivars preferentially accumulated H2O2 and showed only faint NBT staining (Figs 1, 2, 3 & 6; Schraudner et al. 1998), M. sylvestris and two Rumex species exhibited mainly O2–• accumulation (Fig. 6). The sites of ROS accumulation and lesion formation coincided in all these species, suggesting that both types of ROS may be involved in the HR-type cell death, depending on the plant species.
Arabidopsis thaliana with 10 accessions from three continents showed a different behaviour. The prevailing ROS in response to ozone exposure was superoxide, whereas, in addition, four accessions accumulated H2O2. As shown by co-inoculation with SOD and catalase, NBT and DAB staining reflected O2–• or H2O2 accumulation, respectively, in nine of 10 accessions. NBT staining in Sap-0 and DAB staining in Jl-1 was not reduced by the enzyme treatments which may be due to inactivation by inhibitors in these accessions. The most sensitive accessions found in this study showed either O2–• accumulation (Cvi-0; Rao & Davis 1999) or O2–• together with H2O2 (Shokhdara; Figs 3 & 4). The sites, surprisingly, did not coincide in No-0 and Np-0. It could be that different enzymatic sources are responsible for the formation of both types of ROS, being located at different sites in the leaf. The results obtained by Pellinen et al. (1999) using various inhibitors of enzymatic ROS production also pointed to at least two sources for H2O2 production in birch leaves. Alternatively, the levels of SOD activity, converting superoxide into H2O2, or of antioxidant compounds, may exhibit patterns in constitutive or inducible activity and levels over the leaf surface.
At present, it is not clear which mechanism(s) are responsible for the apoplastic ROS accumulation in ozone-treated plants. Work with Arabidopsis and tomato has revealed that ROS production is dependent on a plasma membrane NADPH oxidase (Keller et al. 1998; Amicucci, Gaschler & Ward 1999; Torres, Dangl & Jones 2002). It was implicated by Rao & Davis (1999) that ozone triggers an oxidative burst by activating a NADPH-dependent oxidase in Arabidopsis. Accordingly, recent results from our laboratory show that ozone exposure of tobacco activates two NADPH oxidase homologs at the transcript level (H. Wohlgemuth, C. Langebartels, unpublished results). Alternative mechanisms for apoplastic and peroxisomal ROS production involving amine oxidases, oxalate oxidase and pH-dependent cell wall peroxidases have been proposed (Bolwell 1999; Corpas, Barroso & del Rio 2001). Given the diverse responses of the species reported in this paper, it seems probable that more than one enzymatic source of ROS formation is responsible for the ozone-activated oxidative burst in different species, possibly also in the (distinctly localized) O2–• and H2O2 accumulation in Arabidopsis.
Reactive oxygen species accumulate in ‘burst initiation sites’ in the vicinity of leaf veins
Ozone-induced ROS accumulation was not homogenously distributed over the leaf surface, but concentrated in the periveinal regions in tobacco, tomato, Malva sylvestris and two Rumex species. Since all cells were directly exposed to the stimulus ozone, clusters of periveinal cells (‘burst initiation sites’; Schraudner et al. 1998) may be particularly sensitive to inducing signals or be otherwise predisposed to activate an oxidative burst. Similarly, several responses in plant–pathogen interactions exhibit preference for the veinal system. Rapid death of periveinal cells was observed in the Cf2 resistance gene-mediated localized response of tomato leaves to race-specific elicitors of Cladosporium fulvum (Hammond-Kosack & Jones 1996). ‘Microbursts’ and resultant ‘micro-HRs’ in Arabidopsis were frequently observed adjacent to the leaf veins (Alvarez et al. 1998). It was suggested by Alvarez et al. (1998) that this pattern reflects the greater exposure of periveinal cells to systemic signals and that, when diffusing out of the veins, the concentration is diluted, so that cells near the vascular bundles are more prone to react.
Accumulation of reactive oxygen species in plants exposed to ozone in the field
Until now, studies on ozone-triggered defense responses in plants were only performed in climate chambers and under ozone concentrations exceeding outdoor levels by up to 500% (reviewed in Langebartels et al. 2002). It was therefore asked whether field-grown tobacco or native plant species also accumulated ROS under near-ambient ozone concentrations. Figure 6 demonstrates that ambient summer ozone levels in Germany lead to typical symptoms in the bioindicator tobacco Bel W3. As known for many years, daily recurrent ozone exposure results in the enlargement of existing lesions rather than formation of new spots (Guderian 1985). DAB staining revealed that the borders of the lesions exhibit a ring of H2O2-accumulating cells. This finding suggests that the lesions release signals to the surrounding cells predisposing them for ROS production. It will be interesting to know whether potential amplifying factors such as ethylene, salicylate, NO (Van Camp, Van Montagu & Inzé 1998) or other signals preferentially are produced or accumulate at these sites.
Several native European plants, including M. sylvestris and Rumex species, are as ozone-sensitive as tobacco Bel W3 and exhibit specific leaf injury (HR-type or chlorotic symptoms; Davison & Barnes 1998; Bergmann et al. 1999). As shown in Fig. 6M. sylvestris as well as R. obtusifolius showed marked accumulation of O2–• under 1·7 × ambient ozone levels, but not under ambient ozone or in pollutant-free air. ROS accumulation occurred in symptom-free leaves, and these sites, after 1 to 2 d, turned into lesions in M. sylvestris. As in the case of tobacco Bel W3, the responses were detected at near-ambient ozone levels.
In conclusion, ozone itself or ozone-derived ROS activate an amplification loop of cellular ROS production in sensitive plants. Thereby, ozone erroneously triggers the pathogen-defence pathway leading to HR-type cell death in crop and native plant species. The known ozone activation of all areas of plant defence (Sandermann et al. 1998; Langebartels et al. 2002), now also including the oxidative burst, together with its occurrence during the vegetation period in wide parts of the industrialized world, makes ozone a primary abiotic elicitor and a modulating factor for disease resistance in the field.