Physiological and Yield Responses of Spring Wheat Cultivars under Realistic and Acute Levels of Ozone

: Tropospheric ozone (O 3 ) is widely recognized as the cause of substantial yield and quality reduction in crops. Most of the previous studies focused on the exposure of wheat cultivars to elevated O 3 levels. Our main objectives were to: (i) investigate the consistency of wheat cultivars’ physiological responses across two different realistic O 3 levels; and (ii) compare these physiological responses with those under short acute O 3 exposure. Three commercially available hard spring wheat cultivars bred under semiarid and Eastern Mediterranean conditions were exposed to two different O 3 levels during two consecutive seasons (2016–2018)—36 and 71 ppbv 7 h mean O 3 mixing ratios in open-top chambers. The results were compared to those following short acute O 3 exposure (102.8 ppbv, 7 h mean for 10 days) in a greenhouse. Non-stomatal responses were signiﬁcantly more pronounced than stomatal responses in all cultivars under different levels of O 3 . The speciﬁc cultivar was observed as the most O 3 -tolerant under all experiments. The fact that the same cultivar was found remarkably tolerant to the local semiarid ambient conditions according to other studies and to O 3 exposure based on the present study supports a link between cultivar resistance to drought conditions and O 3 .


Introduction
Tropospheric ozone (O 3 ) is a phytotoxic secondary air pollutant formed by complex photochemical reactions of O 3 precursors such as nitrogen oxides, carbon monoxide, methane, and volatile organic compounds [1]. O 3 has substantial deleterious impacts on both crops and perennial plants [2,3]. Von Schneidemesser et al. [4] highlighted the complex manner in which climate change and O 3 are linked in affecting both human health and crops. O 3 causes damage in plants following its penetration into leaves through the stomata; this leads to oxidative stress, initiating metabolically affluent defense mechanisms and accelerating senescence and reduction in photosynthesis, growth, biomass, and yield [5,6]. Both chronic and acute O 3 exposure are known to affect photosynthesis, induce variation in stomatal responses, decrease in carboxylation efficiency of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and reduce biomass and yield [2].
Furthermore, exposure to O 3 can damage leaves depending on the level and the duration, manifested as foliar injury symptoms on natural vegetation and crop plants [7]. Emberson et al. [8] reported foliar injury and decreased unit leaf area available for photosynthesis under high episodes of O 3 exposure and accelerated senescence under moderate O 3 exposure. Under elevated levels of O 3 , different crop plants also showed visible foliar injury symptoms [9,10].
Wheat (Triticum aestivum L.) is one of the most O 3 -sensitive crops identified to date [10,11]. Chuwah et al. [12] estimated crop damage of up to 20% locally in 2050 due to higher O 3 concentration based on existing exposure-response studies. Schauberger et al. [13] estimated global wheat reductions of about 27% and 39% in Western and Asian wheat, respectively, also causing human health burdens, particularly in Asian countries. Exposure of spring wheat to slightly elevated O 3 concentrations caused a reduction in photosynthesis and yield under glasshouse conditions [14]. Pleijel [15] reviewed 18 wheat genotypes from nine countries that reported an 8.4% reduction in grain yield in non-filtered air compared to charcoal-filtered OTCs after 62% removal of O 3 . Similarly, based on a meta-analysis, Feng and Kobayashi [16] estimated decreases in the wheat yield of~10% and 20% in the present ambient (31-50 ppb) and elevated (51-75 ppb) O 3 levels as compared to charcoal-filtered air-exposed plants. Modeling studies predicted 9-18% global wheat yield losses despite assuming that presently approved air quality legislation will be fully implemented by 2030 [17].
According to Emberson et al. [8], under the present O 3 levels, significant global yield loss of crop plants such as wheat, rice, maize, and soybean is estimated at 2-16%. Based on a meta-analysis, Feng et al. [9] estimated a decrease of~29% in the yield of wheat exposed to average 73 ppb O 3 in open-top chambers (OTCs), growth chambers, and greenhouses (GHs) as compared to carbon-filtered air. The performance of wheat cultivars was also altered by environmental conditions and O 3 required to involve in crop breeding programs [18,19]. Potential interactions between O 3 concentration and plant responses can be expressed by exposure index for O 3 , i.e., accumulated exposure over a threshold of 40 ppb (AOT40), or preferably by phytotoxic ozone dose (POD) and validated by various greenhouse, controlled environment, and field experiments [5,19]. AOT40 or POD were frequently studied by assuming a linear relationship between the O 3 indices, while deviation from linearity was often observed, particularly at low O 3 exposure [10,20]. From a physiological point of view, studying the effects of exposure to low O 3 levels can provide an advantageous insight into the plant's response mechanisms across different realistic O 3 exposure levels, including low levels, and is also useful for assessment requirements [18,21].
The same cultivars would respond fundamentally differently to long moderate chronic exposure versus short and acute exposure to O 3 , raising the need to use robust metrics for O 3 exposure or several different metrics. For instance, Sinha et al. [22] estimated crop-specific exposure-yield functions based on the AOT40 and the mean 7-h day time O 3 mixing ratio (M7) exposure metrics to assess O 3 exposure effects at different growing seasons. Environmental conditions such as drought can also lead to variability in plant response to O 3 exposure; for instance, by affecting stomatal conductance, changes in vapor pressure deficit (VPD) can alter the O 3 uptake [23]. The plant's biochemical mechanism can alter the degree of the O 3 damage through stomatal conductance, which is an additional cause for variation in response of different cultivars to O 3 exposure [2,5].
Therefore, directly including 'sensitivity to ambient O 3 ' as part of the breeding process can significantly improve the response of particular cultivars to specific ambient conditions. Identifying intraspecific alterations in cultivars' responses to O 3 is thus fundamentally important for breeding, and exposure-response experiments are crucial to studying the response mechanisms of different cultivars to O 3 .
We hypothesized that exposing wheat cultivars to two different realistic models-slightly and moderately elevated O 3 levels-and comparing their physiological responses to short acute exposure would demonstrate the fundamental difference in cultivars responses to O 3 . Exposure to different levels of O 3 with different meteorological conditions can also provide important insights for the process of selecting particular wheat cultivars for a specific growing area.
To address this hypothesis, we exposed three wheat cultivars with different phenological characteristics to two different realistic-slightly and moderately elevated-O 3 levels over two entire wheat-growing seasons in near-natural OTCs [10]. We compared the results per cultivar with those from control OTCs (no O 3 enrichment) and those from short-term acute O 3 exposure in the GH. Another key aspect of this study is the potential link between resistance to O 3 and prevailing ambient conditions addressed by focusing on cultivars bred under semiarid conditions. To the best of our knowledge, this is the first report of the effects of O 3 on spring wheat cultivars bred under semiarid and Eastern Mediterranean climate conditions and commercially available for use in Israel.

Experimental Overview
An O 3 exposure-response study was conducted on two experimental platforms: OTCs and GHs (Table 1)

Experimental Plants
Hard spring bread wheat (T. aestivum L.) cultivars Zahir, Gedera, and Ruta, bred and commercially available in Israel, were used for this study. Having no previous information about the response of these cultivars to O 3 , these cultivars were selected according to phenological characteristics [25]. 'Zahir' is a very early-maturing genotype, 'Gedera' is an intermediate-maturing genotype, and 'Ruta' is a late-maturing genotype. A preparatory study was also conducted on four cultivars-Zahir, Omer, Beit Hashita, and Yuval-to test their physiological responses under direct leaf-level O 3 exposure of 65 ppbv (see Section S1 of the Supplementary Information).

GH Experimental Setup
Five replicates per cultivar were exposed to acute doses of O 3 , 78 ± 27 ppbv 24 h mean during the exposure duration (Section 3.1; GH-DE) in one GH section. The same number of replicates of the three cultivars were placed simultaneously in an adjacent GH section with no O 3 enrichment and used as a control (22.1 ± 4 ppbv). Both GH sections were exposed to similar meteorological conditions, which were continuously monitored through the installed sensors (Section 2.3). Wheat seeds were sown in 12 L capacity pots filled with commercially available plant potting mix. Pots were placed under cold and dark conditions until germination and then transferred to the phytotron with 10/16 • C (night/day), 8 h of light. At anthesis, pots were moved from the phytotron to the GH. Plants were irrigated daily using a controlled drip irrigation system and experienced mean T of 20 ± 9.9 • C, relative humidity (RH) 80 ± 12%, and photosynthetically active radiation (PAR) 305 ± 105 µmol m −2 s −1 . Meteorological conditions were recorded during the entire experimental period. Plants in the enriched O 3 section were continuously (24 h) exposed to an acute O 3 mean dose of 78 ppbv for 9 days (Figure 1). Physiological measurements were performed during O 3 exposure (GH-DE) and repeated 5 days after O 3 exposure termination (GH-AE), with plants kept at a low level of O 3 recorded as 22 ppbv (Figure 1).

Experimental Plants
Hard spring bread wheat (T. aestivum L.) cultivars Zahir, Gedera, and Ruta, bred and commercially available in Israel, were used for this study. Having no previous information about the response of these cultivars to O3, these cultivars were selected according to phenological characteristics [25]. 'Zahir' is a very early-maturing genotype, 'Gedera' is an intermediate-maturing genotype, and 'Ruta' is a late-maturing genotype. A preparatory study was also conducted on four cultivars-Zahir, Omer, Beit Hashita, and Yuval-to test their physiological responses under direct leaf-level O3 exposure of 65 ppbv (see Section S1 of the Supplementary Information).

GH Experimental Setup
Five replicates per cultivar were exposed to acute doses of O3, 78 ± 27 ppbv 24 h mean during the exposure duration (Section 3.1; GH-DE) in one GH section. The same number of replicates of the three cultivars were placed simultaneously in an adjacent GH section with no O3 enrichment and used as a control (22.1 ± 4 ppbv). Both GH sections were exposed to similar meteorological conditions, which were continuously monitored through the installed sensors (Section 2.3). Wheat seeds were sown in 12 L capacity pots filled with commercially available plant potting mix. Pots were placed under cold and dark conditions until germination and then transferred to the phytotron with 10/16 °C (night/day), 8 h of light. At anthesis, pots were moved from the phytotron to the GH. Plants were irrigated daily using a controlled drip irrigation system and experienced mean T of 20 ± 9.9 °C, relative humidity (RH) 80 ± 12%, and photosynthetically active radiation (PAR) 305 ± 105 μmol m − ² s −1 . Meteorological conditions were recorded during the entire experimental period. Plants in the enriched O3 section were continuously (24 h) exposed to an acute O3 mean dose of 78 ppbv for 9 days (Figure 1). Physiological measurements were performed during O3 exposure (GH-DE) and repeated 5 days after O3 exposure termination (GH-AE), with plants kept at a low level of O3 recorded as 22 ppbv (Figure 1). Ozone mixing ratio (ppbv)

OTC Experimental Setup
Experiments were conducted in OTCs (control-OTC-CO) and exposed to elevated levels of O 3 (OTC-EO). The OTC was constructed from a transparent perspex sheet. The length of the OTC was 3 m, and the height was adjusted to plant growth using panels of different heights. Initial OTC height was 1 m (from seed sowing until the emergence of the flag leaves). It was gradually increased to 1.5 m when the heading stage began and finally raised to 2 m at full emergence of the heads. All wheat cultivars were grown in replicate rows (n = 5) inside the OTCs with a distance of 15 cm between rows, as per standard agricultural practice. Plants were irrigated through the drip irrigation system according to their water requirement. O 3 was pumped from the CH-ZTW O 3 generator (Guangzhou O 3 Electric Appliance Co., Ltd., Guangdong, China) and mixed with air before injection into the OTCs using a teflon pipe with a flow rate of 60 L m −3 . The main Teflon During season II, cultivars in the OTC-EO were exposed to an average 71 ppbv O 3 (7 h mean) from tillering until maturation, and OTC-CO cultivars were exposed to 31 ppbv O 3 .

O 3 Fumigation and Monitoring
O 3 (7 g h −1 ) was generated from an O 3 generator; pure O 2 was supplied from an O 2 tank to the O 3 generator to achieve the targeted O 3 mixing ratios. O 3 was fumigated over the plant's canopy through a teflon pipe system. Five O 3 sensors were installed and symmetrically distributed over the plants to record the O 3 inside the experimental OTC, and one was installed inside the control OTC. A similar distribution of sensors is mentioned in Section 2.4. The recordings of measured O 3 were collected using a data logger (Model CR800, Campbell Scientific, Logan, UT, USA) at 10 min intervals.
Before installation and weekly during the experiment, each O 3 sensor was calibrated by an O 3 calibrator. T, RH and PAR was measured continuously inside the GH and the OTCs using T and RH sensors (Model 083E-L, Campbell Scientific) and a PAR quantum sensor (Model PQS1, Kipp & Zonen B.V., Delft, The Netherlands), respectively. The data were recorded every 10 min and collected by the logger (Model CR1000, Campbell Scientific). At the OTC experimental setup site, during both seasons I and II, ambient O 3 and meteorological data were recorded from the station, i.e., T, RH and PAR using a T and RH sensor (Model HMP155, Vaisala, Helsinki, Finland) and PAR sensor (Model CM11, Kipp & Zonen), located 20 m from the OTC experimental setup.

O 3 Exposure Indices and Meteorological Parameters
Evaluation of the exposure level of the plants to O 3 concentration used the following O 3 metrics: (i) 12 h daytime (M12; 08:00-19:59) and 7 h daytime (M7; 09:00-15:59); (ii) AOT40 in ppmh, representing the hourly mean O 3 mixing ratios accumulated over a threshold O 3 concentration of 40 ppbv during the daylight hours of the whole growing season, as given by Fuhrer et al. [26].
The ambient 24 h mean O 3 concentrations for the experimental duration were 15 ppbv (season I) and 28 ppbv (season II). Corresponding RHs in seasons I and II were 66 ± 13% and 60 ± 14%, and recorded rainfalls were 299 and 286 mm, respectively.

Physiological Measurements
Gas-exchange measurements (photosynthetic rate; Ps, stomatal conductance; gs, intercellular CO 2 ) were carried out on a randomly selected fully expanded flag leaf using the portable photosynthesis system (LI-6400 XT) with an attached 6400-40 leaf chamber fluorometer (LICOR, Lincoln, NE, USA). Along with standard photosynthetic gas-exchange parameters, the actual photochemical efficiency of photosystem II in saturated light (Fv /Fm ) was logged by the portable photosynthesis system. Before each set of measurements, the assimilation chamber conditions were maintained at 60-70% RH, leaf T was set at 20 • C, and CO 2 concentration was maintained at 400 ppm in the leaf chamber. The flow rate was set to 300 µmol s −1 , and the flag leaf was illuminated with a photosynthetic photon flux density of 1500 µmol m −2 s −1 via LED light source present in the internal light chamber.
For the GH experiments, measurements were performed during and after O 3 exposure (GH-DE and GH-AE, respectively) and on the respective controls ( Figure 1), with three replications per treatment. In the OTC experiments, measurements were carried out on five replicates of each cultivar from both control and O 3 -enriched OTCs. Measurements on flag leaves were carried out according to the phenological development of the cultivars, at heading, anthesis, and grain filling stages ( Table 2).
The response curves of CO 2 assimilation rate (A) to intercellular CO 2 (Ci) concentration, namely the A/Ci curves, were recorded for the flag leaves using an automatic A/Ci curve program with a portable gas exchange system. The steady-state rate of net photosynthesis (A) was recorded at 11 concentrations of external CO 2 : 400, 300, 200, 100, 50, 400, 600, 800, 1000, 1200, and 1400 ppm. There was a minimum 120 s and a maximum 180 s wait time for the instrument to reach the required CO 2 concentration under saturating irradiance of 1500 µmol m −2 s −1 at a leaf T of 20 • C and RH of 60-70%. A/Ci response curves data were recorded automatically four times to ensure data stability. The maximum carboxylation rate allowed by Rubisco designated Vcmax and the electron transport rate for RuBP regeneration and designated as J were determined by subjecting A/Ci curve data, which were obtained for each flag leaf using curve fitting according to Sharkey et al. [27]. The program followed the model proposed by Farquhar et al. [28] suggesting an A/Ci response curve of photosynthetic CO 2 assimilation versus CO 2 inside the leaf based on the notion of a calculated CO 2 partial pressure inside a leaf.

Yield Data Measurement for OTC Experiments
Plants were sampled at maturity from each replicated row (n = 5) of cultivars excluding the plants at the edge of the chamber (0.25 m from each side) for both control and O 3enriched OTCs. Plants were oven-dried at 60 • C up to a constant weight to determine biomass and yield components.

Statistical Analysis
Both experiments, GH and OTC, were randomly designed as a nested model assigning the main plot to the O 3 treatment and the subplot to the cultivar. For each experimental condition (GH and OTC), analysis of variance (ANOVA) for each variable was performed with factors 'O 3 ' and 'cultivar'. O 3 and Cv × O 3 effects could be potentially confounded due to variations in soil properties across the two chambers, although such variations are not probable considering soil homogeneity in the study area and the short distance between the two OTCs (~1 m).
The study area was selected to ensure homogeneity soil properties and irrigation level across the experiment and the control OTCs, which were distanced 1 m from one another with no mutual shading. No previous ozone exposure experiment was performed in the research area. Therefore, a nested model was selected for statistical analysis showing the effects of O 3 and cultivar within O 3 , Cv(O 3 ). For the OTC experiment, statistical analysis was conducted after binning the physiological data and grouping according to the phenological stage of the cultivar (See Table 2). The Student's t-test was used to compare each cultivar's assessed parameters between controls and their respective O 3 treatment across phenological stages in OTC and GH experiments. Discriminate analysis and bivariate correlation were performed on the physiological parameters data derived from the percent difference between O 3 and control for the joint GH and OTC data set. All data are presented as mean ± standard error of the mean (mean± SE) of cultivar replicates with a significance level set at p ≤ 0.05. Statistical analysis was performed with JMP 13 software (SAS Institute, Cary, NC, USA).

OTC Experiment
Meteorological data (24 h means) for T, RH, VPD, and global solar radiations are shown in Figure 2. Ambient air showed higher T and VPD for season II than for season I, from emergence until maturity of the cultivars, particularly up to the end of the grain-filling stage. Environmental conditions inside and outside the OTC suggested that plants inside the OTC were experiencing near-natural conditions (see Figure S4). Table 3 presents the data of O 3 mixing ratios in the OTCs and ambient air for both seasons. Higher concentrations of O 3 were recorded in season II compared to season I, associated with higher AOT40 for the former. Cultivars experienced slightly different O 3 exposure according to their phenological development (Figure 3). Data of the physiological measurements were grouped according to the plant phenological stage ( Table 2). The running averages of the O 3 mixing ratios and AOT40 for each cultivar during heading, anthesis, and grain filling are shown in Figure 3B. Cultivars that had earlier phenological development (and flag leaf sheath opening) experienced lower O 3 , especially at the heading stage, as seen, for instance, for 'Zahir' in Figure 3B. However, note that AOT40 in season II was~15-fold higher than in season I.

Foliar Injury
Foliar injury symptoms appeared within 2 days of continuous exposure of plants to high O3 in the GH (GH-DE) (Figure 4). After terminating the O3 exposure (GH-AE), foliar injury symptoms persisted in all cultivars but did not extend to necrosis. Damage

Heading Anthesis
Grain filling

Foliar Injury
Foliar injury symptoms appeared within 2 days of continuous exposure of plants to high O 3 in the GH (GH-DE) ( Figure 4). After terminating the O 3 exposure (GH-AE), foliar injury symptoms persisted in all cultivars but did not extend to necrosis. Damage appeared on the adaxial surface of the leaves as small pale-yellow blotches between the veins, which is a typical response to exposure to O 3 [29]. In season I, plants exposed to slightly higher levels of O 3 in the OTC did not exhibit significant visible damage. In season II, plants under moderately high O 3 exposure from their initial growth stage in the OTC showed foliar injury symptoms from anthesis in all cultivars (Figure 4).

GH Experiment
ANOVA showed statistically significant differences in physiological parameters according to O3 exposure and cultivar effects for GH-DE and GH-AE ( Figure 5). The physiological measurements are shown individually for each cultivar in Figure 5. Ps and Fv′/Fm′ decreased, whereas Ci increased and gs exhibited varied responses between the O3-enriched GH and the corresponding control for both GH-DE and GH-AE. The least reduction in Ps was observed in 'Zahir' consistently for GH-DE and GH-AE, whereas 'Ruta' showed the largest reduction under both GH-DE and GH-AE (Figure 5a). A reduction of 15.3% in gs was observed without a cultivar effect for GH-DE. For GH-AE 'Gedera' and 'Zahir' showed increases of 7% and 8% in gs, respectively, whereas a reduction of 19% was observed in 'Ruta' (Figure 5b). An increment in intercellular CO (Ci) was observed in all cultivars under O3 enrichment compared to their controls during O3 exposure (GH-DE) and afterward (GH-AE) (Figure 5c). The actual photochemica efficiency of PSII in saturated light designated Fv′/Fm′ decreased significantly in al cultivars, both during O3 stress and after recovery. Maximum reductions of 22% and 18% were observed in 'Ruta' in GH-DE and GH-AE compared to control plants, respectively (Figure 5d). The A/Ci response curves to O3 exposure during GH-DE and GH-AE for the three cultivars are shown in Figure S2. Under both GH-DE and GH-AE, the photosynthetic rate started saturating at 600 μmol mol −1 in all cultivars. Vcmax was reduced during and after the O3 exposure in all cultivars; however, insignificant reductions in J were recorded except Gedera at GH-DE ( Figure 6). During exposure (GH-DE), Vcmax of 'Gedera', 'Ruta', and 'Zahir' decreased by 18.5%, 28.2%, and 15.4% respectively. However, comparatively lower reductions, 17.7%, 16.6%, and 9.7%, were observed after the termination of O3 exposure in 'Gedera', 'Ruta', and 'Zahir', respectively ( Figure 6).  Figure S2. Under both GH-DE and GH-AE, the photosynthetic rate started saturating at 600 µmol mol −1 in all cultivars. Vcmax was reduced during and after the O 3 exposure in all cultivars; however, insignificant reductions in J were recorded except Gedera at GH-DE ( Figure 6). During exposure (GH-DE), Vcmax of 'Gedera', 'Ruta', and 'Zahir' decreased by 18.5%, 28.2%, and 15.4%, respectively. However, comparatively lower reductions, 17.7%, 16.6%, and 9.7%, were observed after the termination of O 3 exposure in 'Gedera', 'Ruta', and 'Zahir', respectively ( Figure 6).

Physiological Measurements in OTC Experiments during Seasons I and II
In season I, ANOVA results showed a statistically significant reduction in Ps due to O3 exposure. However, statistically insignificant responses across cultivars at all phenological stages were observed for Cv(O3). Variations in the gas-exchange responses (Ps, gs, Ci, and Fv′/Fm′) of each cultivar exposed to a higher level of O3 in OTC-EO vs. OTC-CO were noted in both seasons I and II (Figure 7a-d). More statistically significant ANOVA results were observed in season II vs. season I (Figure 7). The largest reduction in Ps was observed at the grain-filling stage for both seasons in all cultivars (Figure 7a,e).
A statistically significant reduction in gs was found in all cultivars; during season I, this was limited to the heading stage, and during season II, it occurred at all phenological stages (Figure 7b). Except for the O3 effect at the heading and the anthesis stages, statistically significant variations in Ci across cultivars and O3 were observed at all phenological stages during season II (Figure 7c). Insignificant reductions were observed in the actual photochemical efficiency of PSII in saturated light (Fv′/Fm′) during season I (Figure 7d). In season II, statistically significant reductions in Fv′/Fm′ were observed at all phenological stages following the trend 'Gedera' > 'Ruta' > 'Zahir' (Figure 7d). The variations in A/Ci for each cultivar under OTC-EO and OTC-CO in both seasons are shown in Figure S3 a,b. ANOVA results showed significant reductions in Vcmax and J due to O3 at all phenological stages in seasons I and II, except for the cultivar effect at the heading stage ( Figure 8). The trends of reduction in Vcmax in response to exposure to O3 in season I was 'Ruta' > 'Gedera' > 'Zahir' and in season II was 'Gedera' > 'Ruta' > 'Zahir'. J reduced in all cultivars under O3 stress at all phenological stages during both seasons. 'Zahir' consistently showed the least reduction among the cultivars in both seasons, and 'Gedera' showed the largest reduction in season II. However, during season I, at all phenological stages, an indefinite trend of reductions was observed in 'Gedera' and 'Ruta' (Figure 8).

Physiological Measurements in OTC Experiments during Seasons I and II
In season I, ANOVA results showed a statistically significant reduction in Ps due to O 3 exposure. However, statistically insignificant responses across cultivars at all phenological stages were observed for Cv(O 3 ). Variations in the gas-exchange responses (Ps, gs, Ci, and Fv /Fm ) of each cultivar exposed to a higher level of O 3 in OTC-EO vs. OTC-CO were noted in both seasons I and II (Figure 7a-d). More statistically significant ANOVA results were observed in season II vs. season I (Figure 7). The largest reduction in Ps was observed at the grain-filling stage for both seasons in all cultivars (Figure 7a,e).
A statistically significant reduction in gs was found in all cultivars; during season I, this was limited to the heading stage, and during season II, it occurred at all phenological stages (Figure 7b). Except for the O 3 effect at the heading and the anthesis stages, statistically significant variations in Ci across cultivars and O 3 were observed at all phenological stages during season II (Figure 7c). Insignificant reductions were observed in the actual photochemical efficiency of PSII in saturated light (Fv /Fm ) during season I (Figure 7d). In season II, statistically significant reductions in Fv /Fm were observed at all phenological stages following the trend 'Gedera' > 'Ruta' > 'Zahir' (Figure 7d). The variations in A/Ci for each cultivar under OTC-EO and OTC-CO in both seasons are shown in Figure S3a ANOVA results showed significant reductions in Vcmax and J due to O 3 at all phenological stages in seasons I and II, except for the cultivar effect at the heading stage ( Figure 8). The trends of reduction in Vcmax in response to exposure to O 3 in season I was 'Ruta' > 'Gedera' > 'Zahir' and in season II was 'Gedera' > 'Ruta' > 'Zahir'. J reduced in all cultivars under O 3 stress at all phenological stages during both seasons. 'Zahir' consistently showed the least reduction among the cultivars in both seasons, and 'Gedera' showed the largest reduction in season II. However, during season I, at all phenological stages, an indefinite trend of reductions was observed in 'Gedera' and 'Ruta' (Figure 8).  Zahir Gedera Ruta [c] [d] [e] Ci (µmol mol

Yield
In season I, grain, spike, and 1000 kernel weights were significantly reduced by 30%, 9%, and 14%, respectively, in 'Ruta', showing the highest reductions among the cultivars ('Gedera': 22%, 8%, and 14%, and 'Zahir': 16%, 8%, and 7%, respectively). Total biomass and straw biomass were reduced in 'Zahir', 'Gedera', and 'Ruta'; however, straw biomass increased in 'Ruta' (12%) under OTC-EO compared to OTC-CO. In season II, 'Gedera' showed the largest reductions in all yield parameters, followed by 'Ruta' and 'Zahir' (Table 4). ANOVA results showed significance for both factors (O3 and Cv(O3) in total biomass, spike weight, and 1000 kernel weight during both seasons. Grain weight and straw biomass were only significantly influenced by O3 and across cultivars, respectively, in season I. During season II, significant variations in the response to O3 and across cultivars were observed for all yield parameters (Table 4).

Yield
In season I, grain, spike, and 1000 kernel weights were significantly reduced by 30%, 9%, and 14%, respectively, in 'Ruta', showing the highest reductions among the cultivars ('Gedera': 22%, 8%, and 14%, and 'Zahir': 16%, 8%, and 7%, respectively). Total biomass and straw biomass were reduced in 'Zahir', 'Gedera', and 'Ruta'; however, straw biomass increased in 'Ruta' (12%) under OTC-EO compared to OTC-CO. In season II, 'Gedera' showed the largest reductions in all yield parameters, followed by 'Ruta' and 'Zahir' (Table 4). ANOVA results showed significance for both factors (O 3 and Cv(O 3 ) in total biomass, spike weight, and 1000 kernel weight during both seasons. Grain weight and straw biomass were only significantly influenced by O 3 and across cultivars, respectively, in season I. During season II, significant variations in the response to O 3 and across cultivars were observed for all yield parameters (Table 4).

Overall Cultivar Response to O 3 under All Experimental Conditions
For an overall investigation of cultivar responses to O 3 under all experimental setups (OTC and GH), discriminant analyses of physiological trait responses in all the cultivars were performed. Figure 9 represents the discriminant analysis results derived from the reduction (%) in O 3 -enriched vs. control values for both OTC and GH experiments as a biplot that applies physiological parameter distribution by canonical one and two and the cultivar grouping. Among the physiological parameters, Vcmax, Ps, and Fv /Fm' were expressed more in the cultivar-specific responses to O 3 stress, while the response to O 3 exposure in gs was less consistent across cultivars and phenological stages. The main (first factor) physiological parameter showing the highest response was Vcmax, and the second factor showing a significant effect was Fv /Fm . Vcmax and Fv /Fm are non-stomatal factors to which a reduction in Ps under O 3 stress can be attributed.
Bivariate analysis was performed to identify the relationship pattern between the physiological parameters that dominate in the cultivar for both experiments (GH and OTC), as presented in Figure 10. Data used for the bivariate analysis were reductions in the experimental vs. control (in percent) Ps, Fv /Fm , and Vcmax for all cultivars in GH and OTC experiments. Figure 10 indicates that reductions were more pronounced under high-level O 3 exposure (GH-DE) than under slight and moderate O 3 exposure in the OTCs. However, regardless of the different conditions applied for each of the three categories-O 3 level, AOT40, and phenology stage-'Zahir' exhibited the lowest reduction in response to O 3 in all three physiological parameters (Fv /Fm , Ps, and Vcmax) represented in Figure 10. Therefore, it can be concluded that the better performance of 'Zahir' under O 3 stress in terms of higher Ps can be attributed to higher Vcmax and Fv /Fm compared to the other two cultivars.

Physiological Responses under Different Levels of O3 Conditions
During seasons I and II, plants were exposed to slightly and moderately elevated levels of O3, respectively, in OTCs. O3 had detrimental effects on all cultivars (Figure 7)

Physiological Responses under Different Levels of O 3 Conditions
During seasons I and II, plants were exposed to slightly and moderately elevated levels of O 3 , respectively, in OTCs. O 3 had detrimental effects on all cultivars ( Figure 7) but with significantly different cultivar responses in season I vs. season II. This was reflected, for instance, in the different cultivar rankings with respect to reduction in Ps across season I ('Ruta' > 'Gedera' > 'Zahir') and season II ('Gedera' > 'Ruta' > 'Zahir').
It should be emphasized that the AOT40 values used in control OTC varied significantly in the two different seasons. Hence, the ratios between AOT40 values for the experimental and control OTCs were similar for the first season (AOT40(ozone)/AOT40(control) = 25.1, based on AOT40 calculated over M7; Table 3) and the second season (AOT40(ozone)/ AOT40(control) = 26.5, based on AOT40 calculated over M7; Table 3). Despite the significant difference in AOT40 values between the O 3 -enriched OTCs in the two seasons, relative yield and reductions in Ps were similar, which might be considered inconsistent. Comparing our results with previous studies, we noticed that, while a linear regression commonly analyzes relative yield vs. AOT40, this regression is frequently highly scattered [7,10,15].
The comparable relative yields and reductions in Ps across the two seasons may have resulted from the higher temperature in season II, which could significantly facilitate better growing conditions than for season I ( Figure 2). Moreover, the Tmin in season I was lower than during season II ( Figure S5) by 2 • C on average, frequently reaching~2 • C during the night, which caused cold stress that the plants had to recover from during the day. Alternatively, VPD (vapor pressure deficit) might also play a role in affecting relative yield. Higher VPD in season II compared with the season I may lead to lower stomatal conductance and thereby smaller effect by O 3 exposure [5]. Mills et al. [18] estimated that microclimatic conditions such as VPD and temperature significantly affect the wheat yield under long-term chronic O 3 exposure, particularly in warm and dry regions where irrigation may increase potential O 3 uptake.
Our results further demonstrate that the response to O 3 exposure in the different cultivars was initiated at different O 3 levels, which dramatically affected relative photosynthetic performance ( Figure 7) and yield (Table 4). Cultivar-wise physiological responses to O 3 exposure in season II were statistically significant compared to season I (Figure 7). Chronic O 3 exposure in OTCs during both season I and season II led to a reduction in yield that was consistent with the physiology of the cultivars, in line with previous studies [9,18].

Cultivar-Wise Variation and Mechanism of Plant Response to O 3
We observed variation in stomatal responses of cultivars under the three O 3 levels (Figures 5b and 7b). O 3 affects stomatal function by reducing the stomatal conductance rate or by reducing the level of stomatal control [30]. According to Ainsworth et al. [2], long-term chronic O 3 exposure at relatively low concentrations tends to result in lower gs and an increase in Ci. Different wheat cultivars showed reduction in gs under elevated O 3 exposure [31]. O 3 effect on Ps may also reduce the plant's detoxification activity, leading to increased respiration that demands more C for maintenance and repair [8]. Nevertheless, the increase in gs under low O 3 in GH-AE may be due to the repair mechanism induced by antioxidants following the termination of O 3 exposure. A similar response showed by Zahir under OTC-EO in season II at the grain-filling stage (Figure 7b) may also be due to the sluggish stomatal response as a result of moderately high O 3 exposure, which can lead to slow or less effective stomatal control [30]. Previous studies even indicated failed stomatal closure due to acute O 3 exposure [31]. Paoletti and Grulke [30] estimated that O 3 -induced photosynthetic impairment in plants could be attributed to a decrease in carboxylation and electron transport efficiency and direct/indirect effects on stomata. Zapletal et al. [32] observed in Picea albies L. a reduction in gs with increasing O 3 levels, which was attributed to metabolic and cellular responses. Hence, under low and moderate exposure, non-stomatal responses expressed more than stomatal conductance.
In the present study, all three cultivars showed a reduction in Vcmax and J along with an increase in Ci under all experimental conditions (GH and OTCs; Figures 6 and 8, respectively). Therefore, Ps decline was attributed more to the decrease in the Vcmax [8,30].

Physiological and Foliar Injury Responses at Different Phenological Stages
Results from both experiments point to the irregular sensitivity ranking of the phenological stages (heading, anthesis, and grain filling) of cultivars in response to enriched O 3 in the OTCs. The reduction in Ps at all phenological stages was shown by all cultivars during season I and for 'Zahir' and 'Ruta' in season II (Figure 7) without significant change in stomatal conductance and/or decrease in carboxylation capacity [33]. During the grain filling, Ps was lower than at the heading-anthesis stage, in agreement with most previous studies that showed a clear gradual and monotonic reduction in Ps from early to mature stages in crop plants [9,31]. The largest decrease in the grain filling stage of Fv /Fm suggests either accumulation of O 3 damage or higher sensitivity to O 3 during this stage, which may be the cause for the grain yield reduction [9].
No foliar injury symptoms were recorded for any of the cultivars in the season I, even though there were clear reductions in physiological activities and yield with relatively small significant differences compared to season II. Chronic O 3 exposure does not constantly stimulate visible injury symptoms but decreases photosynthesis biomass and yield [2]. Even in the absence of visible foliar injury, O 3 induced damage to the photosynthetic machinery observed in many physiological studies' progressive loss of Rubisco activity and reduction in carbon fixation [34]. This difference across the seasons may reinforce the notion that a similar reduction in Ps and yield across season I and season II can also be attributed to better meteorological conditions for growth for the latter, while the fact that foliar injuries appeared only in season II indicates greater O 3 damage in that season.

Overall Cultivar Response to O 3 under All Experimental Conditions
The discriminant analysis (Figure 9) showed that 'Zahir' is much more tolerant to O 3 stress than the other cultivars in all experimental setups. This can be attributed to a smaller reduction in the non-stomatal factors, for instance, Vcmax and Fv /Fm . A similar effect of O 3 on wheat cultivar variation was observed by Feng et al. [31], who estimated that non-stomatal factors dominate in causing differences in Ps reduction across cultivars. Moreover, during a preliminary study that applied instantaneous exposure of the flag leaf to O 3 , 'Zahir' was also found to have high stomatal conductance, photosynthetic rate, and transpiration rate under O 3 stress compared to the other cultivars and was ranked as the most resistant cultivar (Section S1 and Figure S1). Note further that, while 'Zahir' is an early-maturing variety, Figure 3 indicates a much higher AOT40 for 'Zahir' in season II than for 'Ruta' in the season I, whereas, in both seasons, 'Zahir' appeared to be much more tolerant than 'Ruta' (Figure 7). This notion is also supported by previous studies on wheat cultivars reporting differential sensitivity to O 3 stress majorly attributed to a reduction in Rubisco activity [8,31]. The same trend of cultivar responses to O 3 was observed for both GH and OTC (Figure 10), suggesting two different cultivar-screening approaches for breeding. The first is to expose cultivars to a very high O 3 level for 2-3 days, then measure Ps and Vcmax. The second would involve lower O 3 exposure (realistic levels of O 3 ) applied for a more extended period. The first option should be applied with caution, considering the large reported differences in responses of potted vs. field plants [15] and references therein. The fact that the preliminary experiment (see Section S1 and Figure S1) also pinpointed 'Zahir' as the most resistant cultivar suggests an even faster method for cultivar screening.
Further, this method was applied with a relatively moderately elevated O 3 mixing ratio (~65 ppbv) and solely the flag leaf exposure. Additional study is required to test the suitability of this methodology for cultivar screening. It should be noted that higher resistance to O 3 in 'Zahir' compared to the other cultivars also fits with its yield stability under different growth conditions and tolerance to other stress factors, such as drought [35]. This suggests that 'Zahir' has acquired fundamental properties protecting it from both drought and O 3 stresses.

Conclusions
The main objective of this study was to test the level of consistency of physiological response mechanisms to two realistic-slightly and moderately elevated-O 3 levels. To the best of our knowledge, this study was the first of its kind in the Eastern Mediterranean region, providing essential data for O 3 exposure response in wheat cultivars bred locally. Overall, our study indicates detrimental effects on physiological activities of all cultivars at all O 3 enrichment levels, including the slightly elevated O 3 exposure in the OTC in season I (AOT40 = 0.902 ppmh; M7 = 36 ppbv), although the cultivar-wise variations for season I were statistically less significant than those for season II. While our results clearly indicate significant differences in the physiological responses and the ranking of the cultivars across the two levels of O 3 exposure, surprisingly, both reduction in Ps and yield were similar across the seasons. This highlights the need to study the effect of realistic O 3 levels on wheat cultivars but rigorously take into account potential ambient effects, which can significantly affect both yield and physiology, particularly under low O 3 exposure.
Responses to O 3 at all O 3 exposure levels seemed to be related to reductions in non-stomatal factors. 'Zahir', which is known for its high tolerance to dry and warm conditions, was found to be the most tolerant cultivar to O 3 exposure across all applied experimental conditions, in line with our preliminary study results. This supports a link between cultivar resistance to air dryness or drought and O 3 . The fact that this is not related to the lower amount of O 3 uptake by this cultivar based on AOT40 and gs monitoring indicates efficient cultivar physiological responses and needs a better understanding of the mechanistic linkage between cultivar resistance to drought and O 3 . Furthermore, a better understanding of the mechanism governing cultivar performance under elevated O 3 can provide insight for breeding programs in areas characterized by drought and relatively high levels of O 3 . The similarity in results across all experiments in terms of the non-stomatal response being the most affected factor and 'Zahir' being the most tolerant cultivar indicate that short exposure to O 3 may be a useful methodology for cultivar screening. However, such rapid screening should be further tested and compared with more cultivars under chronic exposure in a field study, considering the notable differences in plant responses to O 3 exposure under controlled potted conditions vs. natural field conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/atmos12111392/s1, Figure S1: Differences in stomatal conductance (gs), net assimilation rate (Ps), and transpiration rate (Trans) of four wheat cultivars due to direct exposure of the flag leaf to O 3 . gs, Ps, Trans were calculated as the difference between their values following an exposure to O 3 Figure S4: Measured meteorological data during the open top chamber (OTC) experiment: Presented are temperature, relative humidity, vapor pressure deficit and photosynthetic active radiation during the season I and II inside the OTCs. Data includes here from all experimental duration (Dec-Apr) for both seasons; In season I, sensors were installed at 31 January 2017 (49 days after emergence (DAE)). For season-II from 11 December 2017 (DAE day 1). Constructed values shows the modified data for season I data from OTC and ambient regression up to the period of sensors without shelter. In season I, PAR sensor was not installed. See more details on the OTC experiment in Sections 2.4 and 3.1.2 in the main text. Figure S5: Ambient daily average Temperature (Tavg) and daily minimum temperature