Isotopic and Water Relation Responses to Ozone and Water Stress in Seedlings of Three Oak Species with Di ﬀ erent Adaptation Strategies

: The impact of global changes on forest ecosystem processes is based on the species-speciﬁc responses of trees to the combined e ﬀ ect of multiple stressors and the capacity of each species to acclimate and cope with the environment modiﬁcation. Combined environmental constraints can severely a ﬀ ect plant and ecological processes involved in plant functionality. This study provides novel insights into the impact of a simultaneous pairing of abiotic stresses (i.e., water and ozone (O 3 ) stress) on the responses of oak species. Water stress (using 40 and 100% of soil water content at ﬁeld capacity—WS and WW treatments, respectively) and O 3 exposure (1.0, 1.2, and 1.4 times the ambient concentration—AA, 1.2AA, and 1.4AA, respectively) were carried out on Quercus robur L., Quercus ilex L., and Quercus pubescens Willd. seedlings, to study physiological traits (1. isotope signature [ δ 13 C, δ 18 O and δ 15 N], 2. water relation [leaf water potential, leaf water content], 3. leaf gas exchange [light-saturated net photosynthesis, A sat , and stomatal conductance, g s ]) for adaptation strategies in a Free-Air Controlled Exposure (FACE) experiment. Ozone decreased A sat in Q. robur and Q. pubescens while water stress decreased it in all three oak species. Ozone did not a ﬀ ect δ 13 C, whereas δ 18 O was inﬂuenced by O 3 especially in Q. robur . This may reﬂect a reduction of g s with the concomitant reduction in photosynthetic capacity. However, the e ﬀ ect of elevated O 3 on leaf gas exchange as indicated by the combined analysis of stable isotopes was much lower than that of water stress. Water stress was detectable by δ 13 C and by δ 18 O in all three oak species, while δ 15 N did not deﬁne plant response to stress conditions in any species. The δ 13 C signal was correlated to leaf water content (LWC) in Q. robur and Q. ilex , showing isohydric and anisohydric strategy, respectively, at increasing stress intensity (low value of LWC). No interactive e ﬀ ect of water stress and O 3 exposure on the isotopic responses was found, suggesting no cross-protection on seasonal carbon assimilation independently on the species adaptation strategy. between net and stomatal conductance for three oak species..


Introduction
Oaks are widely distributed in the Mediterranean basin where drought and air pollution such as tropospheric ozone (O 3 ) are the main limiting factors for productivity [1][2][3]. The diversity of oak species is the result of the adaptation to the environment [4]. A wide phenotypic plasticity helps the

Stable Isotopes
Randomized leaf samples (fully expanded sun-exposed leaves of 4-6th order from the shoot tip) of at least 75-100 mg dry weight (DW) each were gathered at the end of treatments (i.e., on October 15th). Samples were collected from each of the three plants per treatment replicate and joined in a single sample, resulting in three samples for each species per each treatment. Samples were dried and milled in a ball mill.
Stable isotope analyses were performed at the Slovenian Forestry Institute (Ljubljana, Slovenia). An aliquot of each sample (m = 0.200 ± 0.050 mg) was weighed into a tin capsule for C and N stable isotope measurements and silver capsule for O stable isotope measurements using a microanalytical balance Sartorius CPA2P and analyzed with Vario Pyro Cube Elemental Analyzer (Elementar, Germany) coupled with IsoPrime100 Isotope Ratio Mass Spectrometer (IsoPrime, UK).
Both certified reference materials (IAEA-601 and IAEA-602 for oxygen, and USGS40, USGS41, IAEA-N-1, and IAEA-N-2 for carbon and nitrogen) and in-house working standards (acetanilide, benzoide, oak leaves, and cellulose) were used to control the accuracy and precision of the measurements. The analytical precision was <0.1% for carbon and nitrogen, and <0.3% for oxygen, expressed as standard deviation of repeated measurements of in-house working standards (n = 3).
Stable isotope results (δ 13 C, δ 15 N, and δ 18 O) are reported in terms of relative delta (δ) value as a difference between 13 C/ 12 C, 15 N/ 14 N, or 18 O/ 16 O ratio of the sample and international reference material (Vienna PeeDee Belemnite (VPDB), Atmospheric Nitrogen (AIR), and Vienna Standard Mean Ocean Water (VSMOW)), expressed in per mil (% ).

Leaf Gas Exchange
Leaf gas exchange was measured in the same fully expanded sun-exposed leaves (4-6th from the shoot tip) of three plants per replicated plot in each treatment using a portable infrared gas analyzer (CIRAS-2 PP Systems, Herts, UK) at controlled values of CO 2 concentration (380 ppm), leaf temperature (25 • C), photosynthetic photon flux density (PPFD, 1500 µmol m −2 s −1 ), and relative humidity (RH, 40-60%). We determined light-saturated net photosynthetic rate (A sat ) and stomatal conductance (g s ).
Measurements were carried out in two campaigns (June 8-10th and September 27th-October 6th) for all O 3 levels (AA, 1.2AA, and 1.4AA) and an additional campaign (August 6-9th) for two O 3 levels (AA and 1.4AA) on days with clear sky between 9:00 and 12:00 a.m. CET.

Predawn and Midday Leaf Water Potential and Leaf Water Content
Predawn and midday leaf water potential (Ψ predawn and Ψ midday , respectively) were measured on clear-sky days in August with a pressure chamber (PMS Instruments Co., Corvallis, OR, USA) on two to three fully expanded sun-exposed leaves collected from randomly selected shoots (n = 3). Midday leaf water potential was measured between 01:00 and 03:00 a.m. CET under PPDF higher than 1200 µmol m −2 s −1 . Predawn measurements were carried out at 1-2 h before dawn. The leaf water content (LWC) was obtained through the measurement of fresh leaf weight (FW), then dried at 80 • C for 72 h (DW). The leaf water content was calculated as: LWC (%) = (FW−DW)/FW.

Statistical Analysis
The statistical unit was the single plot (n = 3 plots). To account for variability in the samples, a pooled standard deviation was computed from the three replicated plots from each species and experimental conditions. Normality of the population distribution was tested using the Shapiro-Wilk test. The homogeneity of variances was performed with the Levene's test. ANOVA analysis was performed to statistically test the effects of treatment (water and ozone), species, month, and their interaction (significance level, p < 0.05). The effect of O 3 and water stress on the relationship between A sat and g s was tested by analysis of covariance (ANCOVA). Post hoc Tukey test was carried out to define the differences among groups. Relationships between g s and A sat , between LWC and δ 13 C, between g s and δ 18 O were calculated at the species level and tested for significant differences between regression coefficients of water and ozone stress (only significant relationships were shown). Statistical analysis was performed with the OriginPro 8 program (OriginLab Corporation, Northampton, UK).

Results
The seasonal course of A sat and g s differed significantly from June to September-October in each species, with higher values in early summer than in autumn in the deciduous oaks ( Figure 1). δ 13 C, δ 18 O, and δ 15 N were statistically different among species (Table 1), with higher δ 13 C and δ 18 O values in WS than WW plants of Q. robur and Q. pubescens ( Figure 2). δ 13 C and δ 18 O increased in WS seedlings of all species relative to WW seedlings (Table 1, Figure 2). δ 18 O was significantly affected by ozone (in Q. pubescens) and by water stress (in three oak species), while δ 13 C was significantly affected only by water stress (in Q. robur). δ 15 N did not change among treated plants. Q. robur showed significant differences of δ 13 C and δ 18 O between WW and WS in 1.2AA-and AA-treated plants, respectively (Table S1). Plants of Q. ilex showed differences of δ 18 O between WW and WS AA-treated plants (Table  S1). In Q. pubescens, δ 18 O was different between WW and WS in all ozone-level-treated plants, as well as between AA and 1.2AA and 1.4AA in WS plants (Table S1).
The combination of species, water, and O 3 , and of O 3 and water stress resulted in significant effects only for A sat (Table 1). A sat and g s changed between months differently within the three oak species (Figure 1). Light-saturated net photosynthetic rate (A sat ) of Q. robur decreased with season whereas elevated O 3 and drought reduced it in August and September-October; A sat of Q. ilex did not show a clear seasonal change during the experimental period, whereas it was lower in WS than WW plants in September-October; A sat of Q. pubescens reached lower values in WS plants in August and September-October whereas O 3 decreased it in autumn. Stomatal conductance showed lower values in WW + 1.4AA than in WW + AA in August in Q. robur while the WS treatment significantly decreased it in August, although such stomatal closure was not observed in September-October; g s did not show differences between water and ozone plants of Q. ilex (Table S2); in Q. pubescens, g s was lower in WS than WW leaves in August while O 3 did not decrease g s in this species. Light-saturated net photosynthetic rates (Asat) and stomatal conductance (gs) of leaves of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity; WS, 40% field capacity) in June, August, and September-October. The bars represent mean ± S.E. (n = 3 plots). Different letters show significant differences among treatments (p < 0.05, Tukey test) within each month for each species. In addition, different capital letters inside parentheses on the x axis denote significant differences among measured months (p < 0.05, Tukey test) in each species. N.D. denotes no data available.

Figure 1.
Light-saturated net photosynthetic rates (A sat ) and stomatal conductance (g s ) of leaves of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O 3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity; WS, 40% field capacity) in June, August, and September-October. The bars represent mean ± S.E. (n = 3 plots). Different letters show significant differences among treatments (p < 0.05, Tukey test) within each month for each species. In addition, different capital letters inside parentheses on the x axis denote significant differences among measured months (p < 0.05, Tukey test) in each species. N.D. denotes no data available. Predawn water potential was higher in WW than WS plants of the three oak species (Figure 3; Table S1). Midday water potential of Q. ilex reached higher values in 1.2AA plants than AA and 1.4AA plants ( Figure 3; Table S1). Q. pubescens showed that Ψ midday was higher in 1.2AA plants than others, it was different between AA and 1.2AA of WW and WS plants, and between 1.2AA and 1.4AA of WW plants ( Figure 3; Table S1). LWC of Q. ilex reached higher values in AA and 1.2AA plants than in 1.4AA (equally in WW and WS) plants ( Figure 3); in detail it was different between AA and 1.4AA, and 1.2AA and 1.4AA in both WW-and WS-treated plants (Table S1). In Q. robur, LWC was lower in WS of 1.4AA plants than other conditions (Table S1). LWC of Q. pubescens was lower in WS plants, both 1.2AA and 1.4AA, than WW plants, and differences were found between AA and 1.2AA in WS, and between AA and 1.4AA in WW plants ( Figure 3, Table S1).
The slope of the relationship between A sat and g s was significantly affected by the combined treatments of ozone and water irrigation in the three oak species (Figure 4, Table S3). Water irrigation increased the slope especially in AA for all three species. On the other hand, 1.4 × AA O 3 exposure increased the slope just in WW-treated Q. pubescens leaves.
Significant relationship between LWC and δ 13 C was found in Q. robur and Q. ilex ( Figure 5). Q. robur showed a reduction of δ 13 C at increasing LWC, while Q. ilex increased δ 13 C at increasing LWC ( Figure 5). Q. pubescens did not show relationship between LWC and δ 13 C. A significant relationship between g s and δ 18 O was found in all species, and did not differ with water and ozone levels ( Figure 5). . Predawn leaf water potential (Ψpredawn), midday leaf water potential (Ψmidday), and leaf water content (LWC) of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity, white bars; WS, 40% field capacity, grey bars). Different letters show significant differences between water levels at same O3 level, different letters inside parentheses show significant differences between ozone levels at same water treatment (p < 0.05, Tukey test, n = 3 plots) in each species. N.D. denotes no data available. . Predawn leaf water potential (Ψ predawn ), midday leaf water potential (Ψ midday ), and leaf water content (LWC) of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O 3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity, white bars; WS, 40% field capacity, grey bars). Different letters show significant differences between water levels at same O 3 level, different letters inside parentheses show significant differences between ozone levels at same water treatment (p < 0.05, Tukey test, n = 3 plots) in each species. N.D. denotes no data available. Significant relationship between LWC and δ 13 C was found in Q. robur and Q. ilex ( Figure 5). Q. robur showed a reduction of δ 13 C at increasing LWC, while Q. ilex increased δ 13 C at increasing LWC ( Figure 5). Q. pubescens did not show relationship between LWC and δ 13 C. A significant relationship between gs and δ 18 O was found in all species, and did not differ with water and ozone levels ( Figure  5). Relationship between light-saturated net photosynthetic rate (Asat) and stomatal conductance (gs) of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, and 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity; WS, 40% field capacity). Pooled data from June to September-October were used. Asterisks show the significance of an ANCOVA: * p < 0.05. Different capital letters denote significant differences of the slopes among the treatments (p < 0.05). All regressions were statistically significant (p < 0.05). The values of slope, y-intercept, and determination coefficient (R 2 ) are listed in Table S3. Relationship between light-saturated net photosynthetic rate (A sat ) and stomatal conductance (g s ) of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O 3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, 1.2AA, and 1.4AA, respectively) and two levels of water irrigation (treatment: WW, 100% field capacity; WS, 40% field capacity). Pooled data from June to September-October were used. Asterisks show the significance of an ANCOVA: * p < 0.05. Different capital letters denote significant differences of the slopes among the treatments (p < 0.05). All regressions were statistically significant (p < 0.05). The values of slope, y-intercept, and determination coefficient (R 2 ) are listed in Table S3.

Ozone Effects
Light-saturated photosynthesis and stomatal conductance differed among oak species (Table 1), showing higher values in the two deciduous species than in the evergreen one (according to [34]), which may result in high stomatal ozone uptake and high ozone damage in deciduous trees [31,35]. Our results confirm that deciduous English oak (Q. robur) is more sensitive to O3 than downy oak (Q. pubescens) and evergreen holm oak (Q. ilex) [28,31], as Q. robur showed lower values of Asat when exposed to the highest ozone levels (1.4AA). Ozone exposure usually reduces stomatal conductance [36,37] (Figure 1). This was confirmed in WW O3-exposed Q. robur leaves in August. However, in autumn, even when Asat decreased, gs remained high in Q. robur, implying a less efficient stomatal control after O3 exposure (i.e., stomatal sluggishness) [38][39][40]. On the other hand, O3 impact on stomatal conductance was not marked in Q. ilex and Q. pubescens throughout the experiment,

Ozone Effects
Light-saturated photosynthesis and stomatal conductance differed among oak species (Table 1), showing higher values in the two deciduous species than in the evergreen one (according to [34]), which may result in high stomatal ozone uptake and high ozone damage in deciduous trees [31,35]. Our results confirm that deciduous English oak (Q. robur) is more sensitive to O 3 than downy oak (Q. pubescens) and evergreen holm oak (Q. ilex) [28,31], as Q. robur showed lower values of A sat when exposed to the highest ozone levels (1.4AA). Ozone exposure usually reduces stomatal conductance [36,37] (Figure 1). This was confirmed in WW O 3 -exposed Q. robur leaves in August. However, in autumn, even when A sat decreased, g s remained high in Q. robur, implying a less efficient stomatal control after O 3 exposure (i.e., stomatal sluggishness) [38][39][40]. On the other hand, O 3 impact on stomatal conductance was not marked in Q. ilex and Q. pubescens throughout the experiment, suggesting a low stomatal reactivity. An effect of ozone was also detected on Ψ midday , suggesting higher water stress, especially in Q. pubescens at the 1.2AA ozone level (Figure 3), due to O 3 exposure in the daytime at the highest evapotranspiration demand. This response is in contrast with the observed decline of g s under O 3 , but in agreement with stomatal sluggishness (i.e., an imperfect or delayed closure of O 3 -exposed stomata) [36]. In addition, O 3 may modify root anatomy, decreasing the water flux of roots in consequence with the reduced water supply to aboveground parts in the target oak species [29]. Interestingly, the overall effect of O 3 on the responses to water deficit was small in the short term (with a slight decline of g s and Ψ midday ) but significant at the end of the experiment (δ 18 O increased), while the overall effect on the carbon balance was highly significant in the short term (A sat decline) but carbon isotope responses were not significant at the end of the experiment (δ 13 C). Similar findings were observed in O 3 -exposed Fagus sylvatica leaves [41,42], highlighting a seasonal variability of O 3 effects on A sat and g s . In theory, stomatal closure may lead to a decrease in the intercellular CO 2 concentration (C i ) [43]. However, this may be compensated by the accompanying decrease in photosynthetic rate due to elevated O 3 , which results in increases of C i [36]. Even more interestingly, such responses were not species-specific in spite of the different O 3 and water deficit sensitivity, suggesting that the three oak species modulated stomatal responses to partly compensate the O 3 -induced seasonal losses in carbon assimilation.

Water Deficit Effects
The three oak species showed seasonally contrasting patterns in their A sat response to water deficit. However, the reduction of photosynthesis of oaks was observed in drought-exposed plants in September-October, highlighting the influence of environmental constraints on tree carbon assimilation [44]. Also, the stomatal conductance was reduced by low water availability in Q. robur and Q. pubescens, whereas g s of Q. ilex was maintained at the same level from June to September-October. Higher sensitivity of gas exchange to water deficit was observed in deciduous oaks than in the evergreen Q. ilex, which is consistent with the previous finding [45]. A significant increase of the slope in the relationship between A sat and g s across the irrigation regimes was found in three oak species, suggesting an increased intrinsic water use efficiency under water stress conditions (Figure 4). The WS-treated Q. robur leaves had a high slope (Table S3), indicating that water deficit intensively induced stomatal closure in this species, which results in a reduced supply of CO 2 to the carboxylation sites [46,47]. For all oak species, δ 18 O differed between WW-and WS-treated plants exposed to AA ozone level, resulting in high δ 18 O values from water-limiting conditions, and reduced total canopy transpiration. Water stress generally limits the uptake and transfer of N into the upper plant parts, resulting in an increased δ 15 N [48]. However, no response of δ 15 N in Q. ilex and Q. pubescens suggests that the responses of g s were not severe, in contrast with what was observed in crops [49]. However, in Q. robur, δ 15 N was affected by ozone exposure in well-watered plants (WW), with higher values in 1.2AA and 1.4AA than AA WW plants, indicating a sensitivity to environmental constraints other than growth conditions (Figure 2, Table S1).

Combined Responses
To the best of our knowledge, this is the first study about effects of these combined stressors (namely water deficit and ozone exposure) on isotope signature in the discrimination of adaptation strategies in three species from the same genus (Quercus sp.). Although stable isotope ratios are considered a reliable proxy of plant stress condition and oak species were discriminated on the basis of δ 13 C for water stress, the combination of water and O 3 stress did not result in any significant interaction on the isotope signatures. These results confirmed the previous study where the carbon isotopic discrimination was observed in relation to water deficit, but not to ozone in seedlings of Fagus sylvatica, Quercus robur, and an ozone-sensitive Populus clone [50]. The only significant ozone x water interaction was on the responses of A sat . It has been reported that fast-growing plant species, namely poplar, with high water requirements are more susceptible to ozone and drought stress [50]. In midsummer and autumn, water stress and O 3 negatively affected A sat in Q. robur (Figure 1). On the other hand, A sat of Q. pubescens was reduced by ozone and by water treatment, although water stress had stronger negative effects than ozone stress (Figure 1). The photosynthetic rate of Q. ilex was reduced by water stress in autumn, showing seasonal plasticity ( Figure 1). However, drought did not induce decoupling in the oak response of A sat to g s , and minor effects of ozone were observed, suggesting a resistant plant behavior to a changing environment. Such behavior might be a potential mechanism of oak species to adapt to disturbances such as drought and O 3 .
The isotope signature highlighted an isohydric behavior of Q. robur, where δ 13 C and δ 18 O were high in response to water stress ( Figure 2; Table S1). In detail, δ 13 C was significantly affected by water stress, while δ 18 O was significantly affected by ozone and by water stress (Figure 2; Table S1). The species showed natively different sensitivity of δ 13 C to LWC in Q. robur (negative correlation) and Q. ilex (positive correlation) ( Figure 5), although it did not show differences in response to water stress and ozone levels of the experiment. Therefore, the 40% reduction of irrigation in the experiment determined a mild stress to be detectable through LWC, which is representative of realistic field conditions. Overall, δ 13 C decreased under water stress and δ 18 O increased under ozone stress, while δ 15 N did not respond to water and ozone stress. Q. robur and Q. pubescens exhibited a more "conservative" water use strategy, characterized by tight stomatal control of transpiration in order to maintain high water use efficiency (according to [50,51]). On the other hand, Q. ilex showed a low variability in stomatal conductance in comparison to the other species, suggesting an anisohydric behavior to maintain stomata open under water deficit [52].

Conclusions
Responses of photosynthesis to water stress were species-specific, with Q. pubescens and Q. robur more sensitive than Q. ilex, suggesting different adaptation strategies of deciduous and evergreen species. Effects of O 3 on physiological traits were lower than those induced by water stress. In detail, the significant interactions of plant water status and O 3 stress on photosynthesis were as expected, that is, water deficit reduced the negative impacts of O 3 . Surprisingly, the effect on photosynthesis did not translate into effects on the seasonal carbon assimilation, that is, δ 13 C did not show a significant response to the combination of water and O 3 stress, suggesting adaptation strategies to overcome the growing season. However, δ 13 C, δ 18 O, and δ 15 N were different among species, with higher δ 13 C and δ 18 O values in WS than WW plants of Q. robur and Q. pubescens, whereas δ 15 N did not change among treated plants. Different acclimation strategies to cope with stress conditions were found, through the different sensitivity of δ 13 C to LWC in Q. robur (negative correlation) and Q. ilex (positive correlation). However, no significant isotopic responses were found to discriminate combined treatments and to differentiate response strategy across the studied oak seedlings. These results warrant further isotopic studies over longer time periods and wider ranges of plant species, in order to understand the carbon and water responses of vegetation to a changing environment and different levels and combinations of environmental constraints.