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Article

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

by
Claudia Cocozza
1,*,
Elena Paoletti
2,
Tanja Mrak
3,
Saša Zavadlav
3,
Tom Levanič
3,
Hojka Kraigher
3,
Alessio Giovannelli
2 and
Yasutomo Hoshika
2
1
Department of Agriculture, Food, Environment and Forestry, University of Florence, Via San Bonaventura 13, 50121 Florence, Italy
2
Institute of Research on Terrestrial Ecosystems (IRET), National Research Council (CNR), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy
3
Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenia
*
Author to whom correspondence should be addressed.
Forests 2020, 11(8), 864; https://doi.org/10.3390/f11080864
Received: 27 June 2020 / Revised: 3 August 2020 / Accepted: 6 August 2020 / Published: 8 August 2020
(This article belongs to the Special Issue Relationships between Growth and Water Relations of Trees)
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Abstract

:
The impact of global changes on forest ecosystem processes is based on the species-specific responses of trees to the combined effect of multiple stressors and the capacity of each species to acclimate and cope with the environment modification. Combined environmental constraints can severely affect 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 (O3) 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 O3 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 [δ13C, δ18O and δ15N], 2. water relation [leaf water potential, leaf water content], 3. leaf gas exchange [light-saturated net photosynthesis, Asat, and stomatal conductance, gs]) for adaptation strategies in a Free-Air Controlled Exposure (FACE) experiment. Ozone decreased Asat in Q. robur and Q. pubescens while water stress decreased it in all three oak species. Ozone did not affect δ13C, whereas δ18O was influenced by O3 especially in Q. robur. This may reflect a reduction of gs with the concomitant reduction in photosynthetic capacity. However, the effect of elevated O3 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 δ13C and by δ18O in all three oak species, while δ15N did not define plant response to stress conditions in any species. The δ13C 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 effect of water stress and O3 exposure on the isotopic responses was found, suggesting no cross-protection on seasonal carbon assimilation independently on the species adaptation strategy.

1. Introduction

Oaks are widely distributed in the Mediterranean basin where drought and air pollution such as tropospheric ozone (O3) 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 plants to adapt to chronic environmental stress through the change of morphological and physiological traits [5].
Water shortage affects tree growth through the alteration of metabolism and physiology of primary and secondary meristems [6,7,8]. Reduced photosynthetic carbon assimilation is often reported as a result of stomatal closure under reduced water availability [9,10,11]. In contrast, O3 impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and photosynthesis, thus inducing stomatal closure [12,13]. Although the individual effects of water deficit and O3 pollution are well documented, the interaction has been less studied. A drought-induced stomatal closure may limit O3 uptake thus reducing O3 injury to plants [14]. However, higher effects of drought were found than long-term O3 exposure in seedlings of Q. ilex, where stomata closure induced by drought did not protect from O3 damage [15]. Variations in stable isotope ratios of plant materials allow assessing long-term stress conditions, seasonally, in relation to environmental constraints. Elevated foliar carbon isotope ratio (δ13C) is a proxy of high water use efficiency [16,17]. δ18O values are negatively correlated with stomatal conductance (gs) thus reflecting plant water status [18,19,20,21,22]. δ15N significantly decreases as mean annual rainfall increases [23].
Forecasting the impact of global changes on forest ecosystem processes, such as productivity and community composition, is based on the trees’ species-specific responses to the combined effect of multiple stressors and the capacity of each species to acclimate and cope with the environmental modification. Therefore, combined constraints are highly relevant in the assessment of tree responses in forest ecosystems. The relationship between physiological responses (photosynthetic and stomatal activity) and stable isotope ratios (carbon, oxygen, and nitrogen) allows identifying distinct phenotypic acclimation strategies.
Among the genus Quercus, a wide range of sensitivity to drought exists [24]. Q. robur, deciduous oak, is considered sensitive to drought and some mortality events were reported in the past following dry seasons [25]. Q. ilex, evergreen oak, avoids desiccation by regulating stomatal conductance in response to the increasing of water deficit intensity [26], making this species highly tolerant to drought and well adapted to Mediterranean environments. The drought tolerance of Q. pubescens, deciduous oak, was favored by isohydric stomatal behavior [27] and a less vulnerable hydraulic structure of leaf petioles than Q. robur [24]. Deciduous oaks are usually considered more O3-sensitive than evergreen oaks in responses to O3 [28].
The present study aimed to define the responses of Q. robur, Q. ilex, and Q. pubescens to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration in an open-air facility) and two levels of irrigation (40 and 100% of soil water content at field capacity) through the stable isotope signatures of C, O, and N, and gas exchange. The main hypothesis was that oak species respond differently to O3 and irrigation regimes, by regulating photosynthetic rate and stomatal conductance to preserve leaf water potential and content in response to combined stresses. The results of this study provide novel insights into the impact of a simultaneous pairing of abiotic stresses (i.e., water and O3 stress) on the water and carbon responses of oak species in leaves.

2. Materials and Methods

2.1. Plant Material and Experimental Design

Two-year-old seedlings of potted Q. ilex L., Q. robur L., and Q. pubescens Willd. obtained from nearby nurseries (origins of seedlings: see [29]) were transferred to 10 L pots in autumn 2014 and then raised at the experimental site in Sesto Fiorentino in central Italy (43°48′59″ N, 11°12′01″ E, 55 m a.s.l.), where the experiment was performed from June to October 2015. The seedlings were grown under the combination of 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 (100% and 40% of soil water content at field capacity, denoted as well-watered, WW, and water-stressed, WS, respectively). During rainfalls, the pots were covered by a plastic cover at 1 cm above the pot, so that evapotranspiration from the soil was not affected (data not shown). The volumetric soil water content (SWC) was measured in the root layer (5 cm depth) by EC-5 soil moisture sensors equipped with an EM5b data logger (Decagon Devices, Pullman WA, USA). In WW plants, SWC was close to field capacity (i.e., 0.295 m3 m−3).
Details on the O3 exposure systems are reported in [30]. In short, O3 was distributed by Teflon tubes hanging down from a fixed grid above the trees. Ozone concentrations at plant height (approx. 1 m) were continuously recorded by O3 monitors (Mod. 202, 2B Technologies, Boulder, CO, USA). The daily mean O3 concentrations were 35.1 ± 1.0 ppb, 43.0 ± 1.2 ppb, and 49.0 ± 1.3 ppb in AA, 1.2AA, and 1.4 AA, respectively, over the experimental period [31].
Plots (5 m × 5 m × 2 m) were assigned to each O3 treatment in three replicates, with three plants per each combination of species, water regime, and O3 in each plot. The plant position was changed every month within each plot to eliminate positional effects [32].
Other results from the same experiment are available in [31] (risk assessment), [33] (antioxidants), and [29] (belowground responses).

2.2. 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 (δ13C, δ15N, and δ18O) are reported in terms of relative delta (δ) value as a difference between 13C/12C, 15N/14N, or 18O/16O 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 (‰).

2.3. 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 CO2 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 (Asat) and stomatal conductance (gs). Measurements were carried out in two campaigns (June 8–10th and September 27th–October 6th) for all O3 levels (AA, 1.2AA, and 1.4AA) and an additional campaign (August 6–9th) for two O3 levels (AA and 1.4AA) on days with clear sky between 9:00 and 12:00 a.m. CET.

2.4. Predawn and Midday Leaf Water Potential and Leaf Water Content

Predawn and midday leaf water potential (Ψpredawn and Ψmidday, 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.

2.5. 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 O3 and water stress on the relationship between Asat and gs was tested by analysis of covariance (ANCOVA). Post hoc Tukey test was carried out to define the differences among groups. Relationships between gs and Asat, between LWC and δ13C, between gs and δ18O 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).

3. Results

The seasonal course of Asat and gs 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). δ13C, δ18O, and δ15N were statistically different among species (Table 1), with higher δ13C and δ18O values in WS than WW plants of Q. robur and Q. pubescens (Figure 2). δ13C and δ18O increased in WS seedlings of all species relative to WW seedlings (Table 1, Figure 2). δ18O was significantly affected by ozone (in Q. pubescens) and by water stress (in three oak species), while δ13C was significantly affected only by water stress (in Q. robur). δ15N did not change among treated plants. Q. robur showed significant differences of δ13C and δ18O between WW and WS in 1.2AA- and AA-treated plants, respectively (Table S1). Plants of Q. ilex showed differences of δ18O between WW and WS AA-treated plants (Table S1). In Q. pubescens, δ18O 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 O3, and of O3 and water stress resulted in significant effects only for Asat (Table 1). Asat and gs changed between months differently within the three oak species (Figure 1). Light-saturated net photosynthetic rate (Asat) of Q. robur decreased with season whereas elevated O3 and drought reduced it in August and September–October; Asat 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; Asat of Q. pubescens reached lower values in WS plants in August and September–October whereas O3 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; gs did not show differences between water and ozone plants of Q. ilex (Table S2); in Q. pubescens, gs was lower in WS than WW leaves in August while O3 did not decrease gs in this species.
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 Ψmidday 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 Asat and gs 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 O3 exposure increased the slope just in WW-treated Q. pubescens leaves.
Significant relationship between LWC and δ13C was found in Q. robur and Q. ilex (Figure 5). Q. robur showed a reduction of δ13C at increasing LWC, while Q. ilex increased δ13C at increasing LWC (Figure 5). Q. pubescens did not show relationship between LWC and δ13C. A significant relationship between gs and δ18O was found in all species, and did not differ with water and ozone levels (Figure 5).

4. Discussion

4.1. 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, suggesting a low stomatal reactivity. An effect of ozone was also detected on Ψmidday, suggesting higher water stress, especially in Q. pubescens at the 1.2AA ozone level (Figure 3), due to O3 exposure in the daytime at the highest evapotranspiration demand. This response is in contrast with the observed decline of gs under O3, but in agreement with stomatal sluggishness (i.e., an imperfect or delayed closure of O3-exposed stomata) [36]. In addition, O3 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 O3 on the responses to water deficit was small in the short term (with a slight decline of gs and Ψmidday) but significant at the end of the experiment (δ18O increased), while the overall effect on the carbon balance was highly significant in the short term (Asat decline) but carbon isotope responses were not significant at the end of the experiment (δ13C). Similar findings were observed in O3-exposed Fagus sylvatica leaves [41,42], highlighting a seasonal variability of O3 effects on Asat and gs. In theory, stomatal closure may lead to a decrease in the intercellular CO2 concentration (Ci) [43]. However, this may be compensated by the accompanying decrease in photosynthetic rate due to elevated O3, which results in increases of Ci [36]. Even more interestingly, such responses were not species-specific in spite of the different O3 and water deficit sensitivity, suggesting that the three oak species modulated stomatal responses to partly compensate the O3-induced seasonal losses in carbon assimilation.

4.2. Water Deficit Effects

The three oak species showed seasonally contrasting patterns in their Asat 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 gs 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 Asat and gs 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 CO2 to the carboxylation sites [46,47]. For all oak species, δ18O differed between WW- and WS-treated plants exposed to AA ozone level, resulting in high δ18O 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 δ15N [48]. However, no response of δ15N in Q. ilex and Q. pubescens suggests that the responses of gs were not severe, in contrast with what was observed in crops [49]. However, in Q. robur, δ15N 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).

4.3. 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 δ13C for water stress, the combination of water and O3 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 Asat. 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 O3 negatively affected Asat in Q. robur (Figure 1). On the other hand, Asat 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 Asat to gs, 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 O3.
The isotope signature highlighted an isohydric behavior of Q. robur, where δ13C and δ18O were high in response to water stress (Figure 2; Table S1). In detail, δ13C was significantly affected by water stress, while δ18O was significantly affected by ozone and by water stress (Figure 2; Table S1). The species showed natively different sensitivity of δ13C 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, δ13C decreased under water stress and δ18O increased under ozone stress, while δ15N 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].

5. 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 O3 on physiological traits were lower than those induced by water stress. In detail, the significant interactions of plant water status and O3 stress on photosynthesis were as expected, that is, water deficit reduced the negative impacts of O3. Surprisingly, the effect on photosynthesis did not translate into effects on the seasonal carbon assimilation, that is, δ13C did not show a significant response to the combination of water and O3 stress, suggesting adaptation strategies to overcome the growing season. However, δ13C, δ18O, and δ15N were different among species, with higher δ13C and δ18O values in WS than WW plants of Q. robur and Q. pubescens, whereas δ15N did not change among treated plants. Different acclimation strategies to cope with stress conditions were found, through the different sensitivity of δ13C 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.

Supplementary Materials

The following materials are available online at https://www.mdpi.com/1999-4907/11/8/864/s1: Table S1: ANOVA of δ13C, δ18O, δ15N, Ψpredawn, Ψmidday, and LWC (one-way analysis) of Q. robur, Q. ilex, and Q. pubescens exposed to O3 and water treatments; Table S2: ANOVA (three-way analysis) of net photosynthetic rate (Asat) and stomatal conductance (gs) of Q. robur, Q. ilex, and Q. pubescens exposed to three levels of O3 and two levels of water treatments; Table S3: Summary of regression parameters between light-saturated net photosynthesis and stomatal conductance for three oak species..

Author Contributions

Conceptualization, Y.H. and E.P.; methodology, T.M., S.Z., T.L., H.K.; data curation, Y.H., C.C., A.G.; writing—original draft preparation, Y.H., C.C.; writing—review and editing, Y.H., C.C., E.P., A.G.; project administration, E.P.; funding acquisition, E.P., H.K. (Slovenian part). All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the FP7 Capacities project EUFORINNO (REGPOT no. 315982) funded by the European Union and Research Programme P4-0107 Forest biology, ecology and technology financed by the Slovenian Research Agency. The ozone FACE facility was supported by the Ente Cassa di Risparmio Firenze.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 11 00864 g001 550
Figure 1. 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 (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.
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Forests 11 00864 g002 550
Figure 2. Leaf δ13C, δ18O, and δ15N 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, black squares, solid line; WS, 40% field capacity, white squares, dashed line). 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.
Figure 2. Leaf δ13C, δ18O, and δ15N 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, black squares, solid line; WS, 40% field capacity, white squares, dashed line). 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.
Forests 11 00864 g002
Forests 11 00864 g003 550
Figure 3. 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.
Figure 3. 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.
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Forests 11 00864 g004 550
Figure 4. 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 (R2) are listed in Table S3.
Figure 4. 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 (R2) are listed in Table S3.
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Figure 5. Relationship between leaf water content (LWC) and δ13C (n = 3 plots), and between stomatal conductance (gs) and δ18O (n = 3 plots) of Q. robur (triangles), Q. ilex (circles), and Q. pubescens (squares) exposed to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, black symbols; 1.2AA, grey symbols; 1.4AA, white symbols) (regression functions are shown as solid line) and two levels of water irrigation (regression functions of WW plants are dashed line, and WS plants in dotted line) (regression functions are missing when they are not significant).
Figure 5. Relationship between leaf water content (LWC) and δ13C (n = 3 plots), and between stomatal conductance (gs) and δ18O (n = 3 plots) of Q. robur (triangles), Q. ilex (circles), and Q. pubescens (squares) exposed to three levels of O3 (1.0, 1.2, and 1.4 times the ambient concentration, denoted as AA, black symbols; 1.2AA, grey symbols; 1.4AA, white symbols) (regression functions are shown as solid line) and two levels of water irrigation (regression functions of WW plants are dashed line, and WS plants in dotted line) (regression functions are missing when they are not significant).
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Table
Table 1. ANOVA of Asat and gs (four-way analysis), δ13C, δ18O and δ15N, Ψpredawn, Ψmidday and LWC (three-way analysis), of Q. robur, Q. ilex, and Q. pubescens exposed to O3 and water treatments is reported (p-level: ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant, p ≥ 0.05).
Table 1. ANOVA of Asat and gs (four-way analysis), δ13C, δ18O and δ15N, Ψpredawn, Ψmidday and LWC (three-way analysis), of Q. robur, Q. ilex, and Q. pubescens exposed to O3 and water treatments is reported (p-level: ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant, p ≥ 0.05).
p-Level
Asatgsδ13Cδ18Oδ15NΨpredawnΨmiddayLWC
species**************nsns***
O3****ns**nsns*ns
water************ns***nsns
species X O3*nsnsnsnsnsns***
O3 X water*nsnsnsnsnsnsns
species X water***nsnsnsnsnsns
species X O3 X water *nsnsnsnsnsnsns
month******
species X month******
month X O3ns*
month X water*****
species X month X O3nsns
species X month X water**ns
month X O3 X waternsns
species X month X O3 X waternsns

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Cocozza, C.; Paoletti, E.; Mrak, T.; Zavadlav, S.; Levanič, T.; Kraigher, H.; Giovannelli, A.; Hoshika, Y. Isotopic and Water Relation Responses to Ozone and Water Stress in Seedlings of Three Oak Species with Different Adaptation Strategies. Forests 2020, 11, 864. https://doi.org/10.3390/f11080864

AMA Style

Cocozza C, Paoletti E, Mrak T, Zavadlav S, Levanič T, Kraigher H, Giovannelli A, Hoshika Y. Isotopic and Water Relation Responses to Ozone and Water Stress in Seedlings of Three Oak Species with Different Adaptation Strategies. Forests. 2020; 11(8):864. https://doi.org/10.3390/f11080864

Chicago/Turabian Style

Cocozza, Claudia, Elena Paoletti, Tanja Mrak, Saša Zavadlav, Tom Levanič, Hojka Kraigher, Alessio Giovannelli, and Yasutomo Hoshika. 2020. "Isotopic and Water Relation Responses to Ozone and Water Stress in Seedlings of Three Oak Species with Different Adaptation Strategies" Forests 11, no. 8: 864. https://doi.org/10.3390/f11080864

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