1. Introduction
Ozone (O
3) is a strongly oxidizing pollutant occurring at ground level as a consequence of the interaction between solar irradiation and gases such as nitrogen oxides (NOx), volatile organic compounds (VOCs), and carbon monoxide (CO). Considering its known phytotoxicity [
1] and the global tendency of increasing background concentrations of about 0.3 ppb/y as a consequence of the global increases in temperature and precursors [
2], investigations of the impact of O
3 on vegetation have been strongly prompted during the last few years [
3], especially because this compound may affect plants’ productivity [
4].
Lichens, despite their wide use as bioindicators of air quality [
5], are known for being rather insensitive to O
3 pollution, with lab and field studies showing similar results: a (very) limited influence of O
3 on lichen physiology and biodiversity. In detail, field studies did not find any evidence of a correlation between lichen biodiversity and O
3 concentrations, as evaluated indirectly by the damage occurring in the O
3 supersensitive plant species
Nicotiana tabacum Bel-W3 during summer exposures [
6,
7,
8], while O
3 fumigations at ecologically relevant concentrations under controlled conditions failed to cause relevant injuries to the photosynthetic systems of several lichen species [
9,
10,
11]. These results suggested a very high tolerance of these organisms to environmental O
3 concentrations, with a toxicity threshold higher than the natural concentrations to which they are commonly exposed to.
However, upon increasing the concentrations of O
3 up to levels less or not at all ecologically relevant, the results were different. Fumigations carried out at ca. 1 ppm O
3 indicated contrasting results: a decrease in net photosynthesis in
Parmelia sulcata [
12] and no change in the chlorophyll content of
Cladonia arbuscula, the photobiont of
Cladonia stellaris [
13] or the photosynthesis of
Cladonia rangiformis [
14]. However, acute fumigations at 3 ppm O
3 induced strong physiological and ultrastructural damage in both the photobiont and the mycobiont of the pollution-tolerant species
Xanthoria parietina [
15]. Nevertheless, these authors suggested that the hydration state may play a major role in determining the severity of the damage, thus explaining the ecological insensitivity of lichens to the high environmental levels of O
3 occurring during dry Mediterranean summers.
Similarly to lichens, a limited susceptibility to O
3 of bryophytes has been reported. Fumigations with 150 ppb O
3 for 5 h induced a slight decrease in photosynthesis only in one out of four
Sphagnum species [
16], while fumigations with 70–80 ppb O
3 for 6–9 weeks induced only a modest photosynthetic injury in
Sphagnum recurvum compared with in
Polytrichum commune [
17]. In addition, chronic fumigations of
Sphagnum in open top chambers did not induce reductions in the chlorophyll contents after exposure to 50, 100 and 150 ppb O
3 [
18]. Nevertheless, fumigations carried out for 10 weeks (6 h/d, 4 d/w) with 240 and 320 ppb were shown to cause reductions in the abundance of four mosses species [
19].
Poikilohydric organisms like lichens and mosses are assumed to have higher resistance to gaseous pollutants during the dry state, having a metabolism strictly dependent on their water content [
20], and since during the summer periods of higher O
3 concentrations the metabolism of these cryptogams is largely reduced, they are likely to be less prone to being injured [
6]. Nevertheless, information about the resistance of mosses to O
3 exposure during different hydration states is lacking and in need of clarification.
The aim of this study was to evaluate the tolerance of lichens and mosses to short-term acute O3 fumigation under different hydration states. We hypothesized that stronger damage would occur following exposure under wet conditions. In addition, we checked for the effect of recovery after exposure.
4. Discussion
Short-term acute fumigation with 1 ppm O3 impaired the photosynthetic efficiency of the lichen, with effects being much more evident in the hydrated state and with no recovery after the damage. However, a decreased chlorophyll content was found only in wet samples, suggesting that the mechanisms of action of O3 causing these alterations are not identical. The antioxidant power always being higher in dry samples suggests that a counteraction of the strong oxidizing effect of O3 may be a key factor to protect the integrity of the chlorophyll. Nevertheless, there is clear evidence that O3 sensitivity is dependent upon the hydration state, since—also in terms of photosynthetic efficiency—the damage was much more striking in wet samples, of which the capacity to convert light was almost abolished. The response was similar for the moss, which was, however, less sensitive to O3 stress and showed clear recovery after one week from the fumigation, even if full recovery was achieved only for the chlorophyll content. Unlike the lichen, the moss showed higher values of antioxidant power in wet samples, thus suggesting a role of this latter parameter in protecting the photosynthetic system.
The higher impact of O
3 on wet samples may be determined by the combination of two main factors, the great solubility of this pollutant in water and the fully activated metabolism of both organisms during the hydrated state. Ozone is a very soluble molecule that exhibits a water solubility higher than that of oxygen and a lower affinity for the cellular hydrophobic layers [
26]. After its dissolution in water, O
3 starts its decomposition, leading to the formation of reactive oxygen species (ROS), responsible for all of the oxidizing reactions that further occurred [
27]. Despite ROS being (at low concentrations) natural components of cell metabolism, produced as a result of aerobic respiration and used in several cell processes, exposure to O
3 tends to increase their concentration, generating cellular perturbations, changes in cellular homeostasis and further physiological injuries, following a process known as “oxidative burst” [
28]. The increased metabolism of these cryptogamic species during the hydrated state may have increased their susceptibility to O
3. In fact, being poikilohydric organisms, with metabolic activity largely dependent on the hydration state, lichens and mosses tend to increase their net photosynthetic rate (NPR) under increasing hydration conditions [
29,
30,
31,
32]. However, the combination of these two factors—the O
3 dissolution in water and the activated metabolism during the hydrated state—are probably the main reasons explaining the differential toxicity of lichens and mosses to this pollutant. This behavior, dependent on the hydration state, is known also for other toxic gaseous pollutants such as SO
2, which shows a higher toxicity to lichens and mosses when they are completely wet [
33,
34], in spite of their activated metabolisms and the high affinity of SO
2 for water [
35].
The mechanism of action of O
3 against the chlorophyll pool is complex [
26], but it is generally assumed to be related either to the direct action of ROS on chlorophylls, with subsequent degradation (chlorosis), or to a sort of protection mechanism of the photosynthetic system [
36]. The reduction in the chlorophyll content of wet samples of
E. prunastri after O
3 fumigation is at variance with the results of [
15], which showed a reduction in dry samples of the lichen
Xanthoria parietina, probably determined by the absence of water, which allowed easy O
3 intrusion into the lichen thallus, as noted for CO
2, for which water has an active role in limiting its diffusion inside the lichen cortex [
29,
37]. Lichens may show remarkable differences in O
3 uptake during the dry state [
38], probably depending on differences in their morphology [
39] or tissue structure—as also suggested for vascular plants [
26]—or on other (still unclear) physiological characteristics that also drive their resistance to others pollutants.
The ecologies of
Evernia and
Xanthoria is quite different, the former being hygrophytic and the latter, xerophytic [
40]; thus, they are naturally less hydrated, with a consequent increase in their tolerance to pollutants such as SO
2 [
34,
41]. In spite of their differential ecophysiologies,
Evernia is known to be more hydrophobic than
Xanthoria [
42,
43], the latter having the capacity to hydrate up to 300% of its dry weight vs. the 200% for the former. In addition, once fully hydrated, to achieve full evaporation,
Evernia requires up to 60 min, while
Xanthoria may prolong the hydrated state for a much longer time, up to 180 min [
43]. These marked differences may well explain the different responses of these two species to O
3.
The limited chlorophyll reduction in the wet samples of
Brachythecium is consistent with chronic fumigations (4–6 weeks) carried out on
Sphagnum in open top chambers, during the dry (Summer, RH = 52–80%) and the wet (Autumn, RH = 71–92%) seasons, for which significant reductions in the chlorophyll contents were not found after exposure to a wide range (50–150 ppb) of O
3 concentrations [
18]. These results suggest a limited effect of O
3 on chlorophyll when samples are not under fully hydrated conditions. However, reduction in the chlorophyll content as a consequence of the toxic action of O
3 has been reported for several vascular plants when fumigated under different concentrations, e.g., for soybean (
Glycine max) at 50–130 ppb O
3 [
44], wheat (
Triticum aestivum) at 25–35 ppb O
3 [
45], bean (
Phaseolus vulgaris) at 50–90 ppb O
3 [
46], and
Tilia americana at 120 ppb O
3 [
47].
The acute O
3 fumigation severely impaired the photosynthetic efficiency of both the lichen and the moss, with reductions of ca. 100% in wet samples and ca. 65–80% in dry samples. Similar results were observed for the lichen
X. parietina, for which wet and dry samples showed reductions of ca. 85% and 69%, respectively [
15]. Slight reductions in the photosynthetic efficiency of
Evernia were observed after 6 weeks of exposure to 90 ppb O
3 in a partially continuous hydration state [
38], but were not observed when fumigated for 14 days with 300 ppb of O
3 (4 h/d) under high levels of humidity (RH = 97%) [
48]. The fumigations of four species of
Sphangnum at 150 ppb O
3 for 5 h induced slightly decreased photosynthetic efficiency in one species only [
16], while fumigations of
Sphagnum recurvum and
Polytrichum commune with 70–80 ppb O
3 for 6–9 weeks induced the occurrence of modest photosynthetic injuries only in the former [
17]. The effect of O
3 fumigations on the photosynthetic efficiency of higher plants is well documented, and significant effects were reported in ca. 50% of the studies [
49], e.g., in the summer squash (
Cucurbita pepo), which showed an impairment of this parameter when fumigated for 5 h/d for 5 days with 150 ppb O
3 [
36]. Furthermore, reductions were also observed in
Tilia americana after 28–42 days of exposure to 120 ppb O
3 at 5 h/day [
47], in sugar beet (
Beta vulgaris) and spring rape (
Brassica napus) at 35 ppb O
3 [
50], and wheat (
Triticum aestivum) at 50 ppb O
3 at different growth stages [
51].
After 1 week following fumigation, the dry samples of
Brachythecium showed an almost complete recovery of the chlorophyll content and an increase in photosynthetic efficiency. The occurrence of photosynthetic recovery after O
3 exposure was also observed in some vascular plants after restoration in O
3-free air. In detail,
Solanum tuberosum showed a recovery of its net photosynthesis after 4 days of fumigation at 60–80 ppb O
3 [
52], while
Nicotiana tabacum Bel B (ozone tolerant) did so after 17 h following fumigation with 300 ppb [
53]. In addition,
Quercus ilex and
Q. pubescens showed recoveries in their photosynthesis after 72 h following 4 days of exposure to 9 h/d 300 ppb O
3 [
54].
The analysis of the chlorophyll fluorescence transient confirmed the strong negative effect of O
3 on wet samples and the lower effect on dry samples. Both the lichen and the moss, after the wet exposure, showed a total flattening of the typical OJIP steps, indicating that reduction of plastoquinone A by electrons did not occur, while dry samples showed an intermediate decrease, indicating that the reduction of plastoquinone B by electrons did not occur, confirming the lower susceptibility of the photosynthetic system under the dry state. This behavior was probably determined by the reduced presence of water in these samples (<10%), which may have limited the diffusion of O
3, since its direct effect on the PSII has been excluded [
55]. In fact, when these cryptogamic organisms are dry, most of them put into action strategies to protect their photosynthetic system from photoinactivation [
56], which make them less sensitive to pollutants. In addition, the fast disappearance of O
3, due to its short half-life that ranges from minutes to hours [
27], may play an important role.
The response of the antioxidant activity showed slight increases in the dry fumigated samples of
E. prunastri (+150%) and in the wet ones of
Brachythecium (+128%). The increases in the antioxidant power after the exposure to O
3 may be related to the necessity for the organism to maintain a stable balance between ROS production and elimination to preserve its cellular and metabolic integrity. ROS can work as signals to stimulate cellular defenses but, following the exposure to oxidizing compounds (O
3), their extracellular concentration increases, generating cellular damage. The effects of seasonal O
3 variations on the antioxidant levels of
Picea rubens, clones of
Populus and
Triticum aestivum were reported [
57,
58,
59], and similarly, fumigations with very low O
3 concentrations caused increases in all of the indicators of antioxidant activity during the first 20 min of exposure of
Carica papaya [
60]. In addition, prolonged exposure to 50 ppb O
3 increased the total phenolic content and peroxidase activity of two different wheat cultivars [
51], and fumigations with 32 ppb O
3 for 4 weeks increased the phenolic contents of
Trifolium pratense cv. Bjursele [
61]. The increase in the antioxidant power in dry samples of
E. prunastri may be, and is probably, related to the ability to counteract the (limited) ROS production, irrespective of an inefficient metabolism. The slight increase in the antioxidant power in the wet samples of
Brachythecium was probably due to a first step of the metabolism to counteract the deleterious effects of ROS on the photosynthetic machinery, as suggested by the reductions in photosynthetic efficiency.
After one week of recovery from the O
3 fumigation, dry samples of
E. prunastri still showed a higher antioxidant power than controls, while wet samples showed a decrease; wet samples of
Brachythecium showed a higher antioxidant power compared with control samples. The low antioxidant power of wet
E. prunastri samples may be, and is probably, due to a sort of metabolic stress caused by ROS, as has been observed for higher plants after prolonged exposure to O
3 [
60]. Nevertheless, we may also speculate that the degradation of photobiont cells could have resulted in the formation of molecules with antioxidant properties.
The higher antioxidant expression of wet moss may be due to the already turned on antioxidant metabolism, stimulated to maintain a sort of protection mechanism for the photosynthetic system during its complete recovery [
58] or as a consequence of a physiological stress-memory process that protects the organism from subsequent oxidative exposures, as can occur in higher plants following ecological changes [
62].