The Water Content Drives the Susceptibility of the Lichen Evernia prunastri and the Moss Brachythecium sp. to High Ozone Concentrations

The aim of this study was to evaluate the tolerance of lichens (Evernia prunastri) and mosses (Brachythecium sp.) to short-term (1 h), acute (1 ppm) O3 fumigation under different hydration states (dry, <10% water content, metabolism almost inactive; wet, >200% water content, metabolism fully active). We hypothesized that stronger damage would occur following exposure under wet conditions. In addition, we checked for the effect of recovery (1 week) after the exposure. Ozone fumigation negatively affected the content of chlorophyll only in wet samples, but in the moss, such a difference was no longer evident after one week of recovery. Photosynthetic efficiency was always impaired by O3 exposure, irrespective of the dry or wet state, and also after one week of recovery, but the effect was much stronger in wet samples. The antioxidant power was increased in wet moss and in dry lichen, while a decrease was found for wet lichens after 1 week. Our results confirm that the tolerance to O3 of lichens and mosses may be determined by their low water content, which is the case during the peaks of O3 occurring during the Mediterranean summer. The role of antioxidant power as a mechanism of resistance to high O3 concentrations needs to be further investigated.


Introduction
Ozone (O3) 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 O3 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 O3 pollution, with lab and field studies showing similar results: a (very) limited influence of O3 on lichen physiology and biodiversity. In detail, field studies did not find any evidence of a correlation between lichen biodiversity and O3 concentrations, as evaluated indirectly by the damage occurring in the O3 supersensitive plant species Nicotiana tabacum Bel-W3 during summer exposures [6][7][8], while O3 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 O3 concentrations, with a toxicity threshold higher than the natural concentrations to which they are commonly exposed to.
However, upon increasing the concentrations of O3 up to levels less or not at all ecologically relevant, the results were different. Fumigations carried out at ca. 1 ppm O3 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 O3 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 O3 occurring during dry Mediterranean summers.
Similarly to lichens, a limited susceptibility to O3 of bryophytes has been reported. Fumigations with 150 ppb O3 for 5 h induced a slight decrease in photosynthesis only in one out of four Sphagnum species [16], while fumigations with 70-80 ppb O3 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 O3 [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 O3 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 O3 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.

Experimental
Samples of the lichen Evernia prunastri and the moss Brachythecium sp. were collected at the end of September 2019 in a remote area of the Siena province (Tuscany, Central Italy), far from any local source of pollution. These species were chosen considering their wide use as biological indicators as well as their ease of collection and handling. In the laboratory, samples were cleaned from extraneous material under a stereoscopic microscope using plastic tweezers. After cleaning, one batch of 18 samples was air-dried (residual water <10%) overnight in a climatic chamber at 16 °C and 55% relative humidity (RH), while another batch of 18 samples was fully hydrated (water content up to 250% of dry weight for the lichen, and up to 600% for the moss) overnight in a climatic chamber at 16 °C and 90% RH. Two thirds of the air-dried and fully hydrated samples were fumigated for 1 h at an ozone concentration of 1 ppm using an ozone generator (GPC2000, Ozonosoluzioni, Italy); the remaining third were fumigated for 1 h with O3-free air (control samples). To evaluate the possible recovery of the samples, 50% of the fumigated and control samples were left for 1 week under environmental conditions.

Chlorophyll Content
The total chlorophyll content of the lichen and moss samples was measured using a chlorophyllcontent meter (CCM-300, Opti-Science, Hudson, NY, USA), which indicates the chlorophyll content on a surface basis (mg/m 2 ). The effectiveness of this non-destructive method for the analysis of the chlorophyll content has already been tested [21]. Ten replicates were measured for each experimental unit.

Chlorophyll Fluorescence Analysis
The analysis of the chlorophyll a fluorescence of the photosystem II and the analysis of the chlorophyll fluorescence transient (OJIP) are known reliable techniques to assess the performance of photosynthetic organisms following O3 exposures [22]. In this study, the chlorophyll a fluorescence was investigated using its most common parameter FV/FM, which indicates the maximum quantum efficiency of PS II photochemistry, an indicator of photosynthetic efficiency. The analysis of the chlorophyll a fluorescent transient (OJIP test) was used as a supplementary photosynthetic indicator. Prior to analysis, samples were hydrated in a climatic chamber at 16 °C, 90% RH and 40 μmol/m2/s photosynthetically active radiation (PAR), to obtain their maximal photosynthetic activation. The analysis was run, lighting the samples for 1 sec with a saturating 3000 μmol/m 2 /s red light pulse, using a Plant Efficiency Analyzer (Handy PEA, Hansatech Ltd, Norfolk, UK). Fifteen replicates were measured for each experimental unit.

Total Antioxidant Power
The DPPH assay is a simple and functional method to evaluate the response of the total antioxidant activity after O3 exposure [23]. Samples of ca. 50 mg were homogenized in 1 mL of a solution of ethanol/water (80:20; v/v). Of the homogenate, 100 μL were added to 1 mL of a 100 μM DPPH solution prepared by dissolving 3.9 mg of this compound in 100 mL of methanol/water (80:20; v/v). After the reaction, which occurred in 1 h, samples were read at 517 nm and the results were expressed as % Antiradical Activity (ARA%) according to the formula: where control absorbance = the absorbance of the reagents only. Five replicates were measured for each experimental unit.

Statistical Analysis
Results were expressed as ratios to control samples. Owing to the limited data set, nonparametric statistics were used. Outliers were sought for using the Tukey test. Differences between fumigated and control samples were checked with the Mann-Withney U test. Differences between dry and wet samples, both immediately after fumigation and after recovery, were checked using the non-parametric Kruskal-Wallis ANOVA. The Dunn's test was used for post-hoc comparisons, except for temporal ones, for which the Wilcoxon rank sum test was used. In both cases, a correction for multiple testing was applied according to [24]. All calculations were run using the free software R [25].

Results
The physiological parameters in Evernia sp. and Brachythecium sp. immediately after O3 fumigation, as well as after one week from the latter, are summarized in Table 1. Table 1. Chlorophyll content (Chl), photosynthetic efficiency (FV/FM) and antioxidant power % (ARA) in samples of the lichen (L) Evernia prunastri and the moss (M) Brachythecium sp. exposed dry (D) or wet (W), immediately after (E) 1 h fumigation with 1 ppm O3 or after 1 week of recovery (R). Values are expressed as median ± median absolute deviation of ratios to control values. Values in bold indicate significant (p < 0.05) differences with control values, capital letters indicate significant (p < 0.05) differences between D and W, and lowercase letters indicate significant (p < 0.05) differences between E and R. * values were almost zero and were disregarded for the statistical analysis but were considered significantly different from controls and dry samples. Compared with control samples, 1 h of fumigation with 1 ppm O3 negatively affected the content of chlorophyll only in wet samples, but in moss samples, such a difference was no longer evident after one week of recovery. Photosynthetic efficiency was always impaired by O3 exposure, irrespective of dry or wet conditions, and also after 1 week of recovery, but the effect was much stronger for wet samples. The antioxidant power was increased in wet moss and in dry lichen, while a decrease was found for wet lichens after 1 week.
Differences between dry and wet samples emerged for all the investigated parameters, with wet samples generally showing lower values.
After one week of recovery, moss wet samples showed an almost complete restoration of their chlorophyll content. The photosynthetic efficiency of dry samples almost doubled in moss but did not change in lichens. The antioxidant power decreased in both lichens and mosses.
The fluorescence transient curves ( Figure 1) confirm that remarkable injury occurred to the photobiont of both organisms following O3 fumigations, which persisted after 1 week of recovery. The transient curves flattened out, losing their typical sequence of the OJIP steps as indicated for healthy (control) samples. Fluorescence emission was characterized by a marked reduction in FM values in dry fumigated samples of both organisms, while negligible differences between F0 and FM values were observed for wet fumigated samples, which showed almost flat transients.

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 efficiencythe 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 O3 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, O3 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 O3 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 O3. 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 O3 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 SO2, 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 SO2 for water [35].
The mechanism of action of O3 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 O3 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 O3 intrusion into the lichen thallus, as noted for CO2, for which water has an active role in limiting its diffusion inside the lichen cortex [29,37]. Lichens may show remarkable differences in O3 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 SO2 [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 O3.
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 O3 concentrations [18]. These results suggest a limited effect of O3 on chlorophyll when samples are not under fully hydrated conditions. However, reduction in the chlorophyll content as a consequence of the toxic action of O3 has been reported for several vascular plants when fumigated under different concentrations, e.g., for soybean (Glycine max) at 50-130 ppb O3 [44], wheat (Triticum aestivum) at 25-35 ppb O3 [45], bean (Phaseolus vulgaris) at 50-90 ppb O3 [46], and Tilia americana at 120 ppb O3 [47].
The acute O3 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 O3 in a partially continuous hydration state [38], but were not observed when fumigated for 14 days with 300 ppb of O3 (4 h/d) under high levels of humidity (RH = 97%) [48]. The fumigations of four species of Sphangnum at 150 ppb O3 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 O3 for 6-9 weeks induced the occurrence of modest photosynthetic injuries only in the former [17]. The effect of O3 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 O3 [36]. Furthermore, reductions were also observed in Tilia americana after 28-42 days of exposure to 120 ppb O3 at 5 h/day [47], in sugar beet (Beta vulgaris) and spring rape (Brassica napus) at 35 ppb O3 [50], and wheat (Triticum aestivum) at 50 ppb O3 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 O3 exposure was also observed in some vascular plants after restoration in O3-free air. In detail, Solanum tuberosum showed a recovery of its net photosynthesis after 4 days of fumigation at 60-80 ppb O3 [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 O3 [54].
The analysis of the chlorophyll fluorescence transient confirmed the strong negative effect of O3 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 O3, 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 O3, 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 O3 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 (O3), their extracellular concentration increases, generating cellular damage. The effects of seasonal O3 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 O3 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 O3 increased the total phenolic content and peroxidase activity of two different wheat cultivars [51], and fumigations with 32 ppb O3 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 O3 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 O3 [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].

Conclusions
The fumigation of the lichen Evernia prunastri and the moss Brachythecium sp. with 1 ppm O3 for 1 h highlighted the higher susceptibility of these cryptogamic organisms to this oxidizing pollutant under the hydrated state. An impairment of the photosynthetic efficiency and a reduction in the chlorophyll content were evident. The role of antioxidant power as mechanism of resistance to high O3 levels needs to be better clarified with further investigations. Our results confirm that the tolerance to O3 of lichens and mosses may be determined by their low water content, which is the case during the peaks of O3 occurring during the Mediterranean summer.
Author Contributions: S.L. conceived and designed the experiments; A.V. and G.C. performed the experiments; S.L. and A.V. analyzed the data; M.P. contributed analysis tools; A.V. and S.L. wrote the paper, C.N. supervised the text. All authors have read and agreed to the published version of the manuscript.