Oxidative Air Pollution Ozone And Others Occurrence and phytotoxicity

Oxidant air pollutants, among them the most abundant ozone, are formed in the atmosphere by the free radical driven interaction of precursors (hydrocarbons, nitrogen oxides, molecular oxygen) with solar radiation of wavelengths below 400 nm (Kley et al. 1999). Ozone is the best known of these substances and will be mainly discussed in the following. However, it must be kept in mind that oxidative air pollution is frequently occurring as a cocktail of many potentially toxic substances, for example peroxyacetylnitrate (PAN), aldehydes, peroxides, peroxy radicals and many others. This aggressive mixture of trace gases is commonly called photochemical smog.

The highest peak concentrations of ozone are found in urban regions with high irradiation and a high density of emittents producing the potential precursors (Kley et al. 1999), with the main culprit the burning of petrol products in traffic and industry. But also in remote areas ozone formation from long range transported precursors is facilitated in high elevations due to the high radiation energy which leads to characteristic altitudinal gradients of atmospheric ozone concentrations in alpine regions (Smidt 1996).

Ozone is a strong oxidant highly reactive in aqueous and lipid phases. In water, a variety of reactive products, among them toxic oxygen species, may be formed by spontaneous reactions (Heath and Taylor 1997). In particular hydrogen peroxide (H2O2), the hydroxyl radical (OH"), and the superoxide anion radical have been linked to metabolic changes in plants (Sakaki 1998). Biochemically, ozone is able to oxidize a variety of cell compounds such as sulphydryl groups (also in glutathione) yielding disulphides, double bounds of fatty acids yielding carbonyl groups or hydroperoxides, amino acids and low molecular weight antioxidants, such as ascorbate and tocopherol (Hippeli and Elstner 1996, Mudd 1998).

The potency of ozone to induce acute or hidden injury in plants is well documented (Darrall 1989, Grulke 1999, Kley et al. 1999). Acute injury is mostly present as necrotic spots on the leaves whereas hidden injury leads to a decline in photosynthesis, carbon fixation and, consequently, growth. For some important agricultural plants and forest trees a relation between growth reduction and the ozone dose has been established from results of open-top chamber experiments (Fuhrer et al. 1997).

However, the primary site of ozone attack to leaf tissues and the mechanism of damage induction is still subject to discussion. Due to the reactive potency of ozone the site of the initial reactions would be expected close to the entry point. According to the principles given at the beginning of the present chapter the uptake of ozone to the leaves is governed by the flux through the stomata, since the cuticle is a near absolute barrier for this gas. Due to the reactivity of ozone with cellular chemicals the internal concentrations of this gas has been estimated as near zero by model calculations (Laisk et al. 1989). However, as ozone has no easily traceable isotope the actual dose of the pollutant, i. e. the amount which reacts with chemicals inside the leaf, has not been measured directly. It seems evident that ozone is able to reach the plasmalemma, but it is hardly capable of penetrating deep into the cell itself. The initial site of ozone action may thus be the apoplast, and the lipids and proteins of the membrane (Heath and Taylor 1997).

Although the phytotoxicity of ozone has been unequivocally demonstrated in many cases, the mode of action is still obscure (Mudd 1998). Several possibilities are discussed:

(1) Ozone easily degrades in aqueous phase forming reactive oxygen species (ROS), such as singlet oxygen, the hydroxyl free radical superoxide anion free radical, or hydrogen peroxide (Sakaki 1998). Although plant cells are equipped with highly effective antioxidant defence systems (with prominent participation of glutathione, see Tausz in this volume) which scavenge these toxic molecules, at high ROS production rates the defence capacity may be insufficient. As evidence for the involvement of ROS in ozone damage it has been reported that the pre-treatment of tissues with (superoxide) or (singlet oxygen) scavengers prevented ozone damage. The increase in in leaves treated with near-ambient levels of ozone was shown by EPR-signal analysis (Runeckles and Vaartnou 1997), and the action of singlet oxygen was demonstrated by chemiluminescence (Kanofsky and Sima 1995). Changes in the cell redox state may lead to several reactions, such as enzyme regulations or the activation of defence genes (May et al. 1998).

(2) Direct oxidative attack at the membranes may produce toxic secondary products, which can have a longer life span and may be transported to other locations, such as the chloroplasts. There they can inhibit enzyme activities of the Calvin cycle and lead to the measureable effects, such as the observed decreases in the photosynthetic rates (Heath and Taylor 1997). In combination with light energy this situation will lead to a production of ROS in the chloroplasts according to the scheme presented by Tausz in this volume (scheme in Figure 1 therein). The accumulation of long-lived toxic organic hydroperoxides was found in ozone treated plant tissues (Hewitt et al. 1990).

(3) An interesting interaction of the ozone induced free radical formation with a pathogen defence reaction, the "hyper-sensitive response", possibly induces cell death (Sandermann 1996). The hypersensitive reaction of plant cells towards a pathogen includes the programmed death of the cells adjacent to the infection and thus inhibits the proliferation of the pathogen which needs living cells to feed on. Hydrogen peroxide and other ROS together with plant hormones are involved in the signalling processes leading to this plant response which is described in more detail by Foyer and Noctor in this volume. Ozone or the ozone generated ROS are thought to mimic the signal and 'erroneously' induce programmed cell death responses leading to ozone symptoms (Sandermann 1996).

Glutathione and ozone toxicity

Besides its role in plant sulphur metabolism glutathione is also an important component of the antioxidative system of the cell. Since the direct scavenging capacity of glutathione towards toxic oxygen species under physiological conditions is lower than that of ascorbate or tocopherol (Table 1 in Tausz in this volume), it is rather of importance as a protectant of protein -SH groups and it is essential to regenerate ascorbate from dehydroascorbate in the enzymatic glutathione-ascorbate cycle. The maintenance of the GSH/GSSG ratio is a prerequisite of the normal function of metabolism.

Many references report an increase in leaf glutathione concentrations upon experimental fumigation with ozone in various plants, such as conifers, deciduous trees, and also herbaceous plants (Alscher 1989). A detailed literature review of ozone effects on the antioxidative system of plants is given by Polle (1998). Mostly, changes in GSH concentrations were accompanied by increases in concentrations of other components of the antioxidative system, such as ascorbate or a-tocopherols. Ozone fumigation is also able to induce the enzymes of the glutathione-ascorbate cycle (Polle 1998, Sehmer et al. 1998) which supports a primary role of the antioxidant activity in response to ozone. Changes in the redox state of the GSH/GSSG pools can also be induced by ozone, in particular in sensitive cultivars, and under high ozone concentrations (more than 100 ngm'3). In tobacco, the well-known and extensively studied ozone-sensitive cultivar BelW3 showed a more pronounced oxidation of the GSH redox pool than more resistant varieties and the GR activity declined. Spinach leaves showed a nearly complete oxidation of the GSH pool upon acute ozone stress (Luwe et al. 1993). However, the reports of ozone effects on the glutathione systems are very divergent (Polle 1998) and depend on many factors. As an example, spruce trees showed no response of the antioxidative systems to a long-term fumigation with about double-ambient ozone concentrations without regard of measurable effects on cell divisions (Wonisch et al. 1998, 1999c).

Increased contents of glutathione were also observed in trees subjected to high elevation stress in the field, a stress complex that is composed of high irradiation, low temperatures, and of high atmospheric ozone concentrations. A clear altitudinal gradient of glutathione contents in conifer needles was found in several studies in the Alps (Rennenberg et al. 1997, Tausz et al. 1997). However, in most field studies it is impossible to distinguish between different environmental factors, all of which are potentially inducing oxida-tive stress. Recent field study results on Pinus ponderosa growing along a pollution gradient in the San Bernardino Mountains (Southern California) partly clarified this aspect. In this region, trees at higher elevations are exposed to lower ozone doses, but to a higher natural stress level, whereas ozone load is greatest at the lower elevated plots. The trees under higher natural stress impacts contained more glutathione, i.e. a confirmation of the results found on spruce at elevation gradients in the Alps. On the other hand, the reduction state of the glutathione pool was significantly lower at the high ozone plots (Tausz et al. 1999a), indicating the crucial role of glutathione regeneration under ozone stress (Tausz et al. 1999b, also postulated by Luwe 1996, see below).

Symplast j Ap op last

Symplast j Ap op last

Figure 3, Compartmentation of ozone-induced ROS formation in plant cells. The apoplast contains considerable amounts of aseorbate but not glutathione. Glutathione takes part in ascorbate regeneration in the cytoplasm or in the chloroplast. GSH glutathione, GSSG oxidized glutathione, Asc ascorbate, DHAsc dehydroascorbate, ROS reactive oxygen species.

Figure 3, Compartmentation of ozone-induced ROS formation in plant cells. The apoplast contains considerable amounts of aseorbate but not glutathione. Glutathione takes part in ascorbate regeneration in the cytoplasm or in the chloroplast. GSH glutathione, GSSG oxidized glutathione, Asc ascorbate, DHAsc dehydroascorbate, ROS reactive oxygen species.

Recent experiments were conducted with transgenic plants containing manipulated activities of the glutathione homeostasis metabolism (Lea et al. 1998, Foyer et al. 1998). Poplar trees containing increased concentrations of foliar glutathione or increased activities of glutathione reductase were investigated (Noctor et al. 1997). Exposure of these plants to acute ozone levels demonstrated that constitutively increased pool sizes of glutathione did not ameliorate ozone resistance. Ozone resistance did not correlate with glu-tathione content or redox state in this system. Although elevated GR activity in the chloroplasts prevented the ascorbate pool from being oxidized, this did not lead to increased ozone resistance (Noctor et al. 1997).

As described above, on its way towards the plasma membranes ozone has to pass through the cell wall. The cell wall contains antioxidative compounds able to scavenge reactive oxygen species (Polle 1998). Peroxidases, which are involved in lignin production, represent the main antioxidative capacity in the apoplastic space, but also ascorbate plays a considerable role. The presence of glutathione in the apoplast is negligible. In spruce needles between 0 and 0.1 nmol total apoplastic GSH g"1 needle dry weight were reported by KronfuB et al. (1998): The same needles contained between 0.1

and 0.7 p.mol ascorbate g"1 needle dry weight in the apoplastic space. In other studies, GSH was unmeasurably low in the apoplastic space, such as in beech trees (Luwe 1996) or herbaceous plants (Lyons et al. 1999, in Plan-tago major). These findings confirm that in contrast to ascorbate, glutathione is not an important factor in the 'first line of defence', the antioxidant system of the cell walls (Polle 1998). However, since ascorbate is mainly regenerated through the action of the enzymatic ascorbate-glutathione cycle (Figure 3), cytoplastic GSH is indirectly involved in the apoplastic detoxification capacity of ascorbate. Since the presence of the enzymes of the ascorbate-glutathione-cycle in the apoplast is questionable, the dehydroascorbate formed by oxidation of ascorbate must be transported into the cytoplasm and regenerated therein (Figure 3). A corresponding ascorbate/dehydroascorbate translocation system in the plasmalemma exists (Horemans et al. 1998). This process involves the cytoplasmatic glutathione pool and might require adaptive responses thereof. There are some hints that the regeneration of apoplas-tic ascorbate is a time-consuming process and that therefore an oxidation of the tissue GSH pool may occur with a certain lag phase after ozone episodes (Luwe 1996) or the redox state of the glutathione pool may change upon long-term exposure (Tausz et al. 1999b).

The explanation of such divergent results may be found in the multiple roles glutathione may play in the response to ozone stress. Even in its role as antioxidant directly involved in the scavenging of ROS it is only one part of a complex system, and the specific conditions may require adjustments in the glutathione system (which are measurable as plant responses) or rather in other parts of the defence systems. The glutathione redox pool can play a role in environmental sensing and signalling of stress situations (May et al. 1998). In particular, transient changes of the GSH/GSSG redox state may be the signal for the induction of several defence reactions (Foyer and Noctor, Tausz in this volume). Glutathione is also involved in the pathogen defence reactions (Gullner and Kömives, Foyer and Noctor in this volume) that have much in common with ozone responses of plant cells (Sandermann 1996).

Furthermore, an appealing possibility is the involvement of the GSH re-dox system in long distance signal transduction of pollution stress impacts. Just like sulphurous pollutants, ozone applied to the canopies of spruce trees produces chromosome damages in the root meristems (Müller et al. 2000), often before biochemical or physiological effects in the canopies could be observed (Wonisch et al. 1999c). The signal transduction pathway in these cases is equally enigmatic as upon sulphurous pollutants, but glutathione as an easily transportable compound may be a probable candidate (Zellnig et al. 2000). As for sulphurous gases, for ozone effects a temporal correlation of chromosome responses and total fine root concentrations of glutathione could not be established yet (Wonisch et al. 1999a).

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