Visible Foliar Injury and Ecophysiological Responses to Ozone and Drought in Oak Seedlings

To verify the responses of visible foliar injury (VFI), we exposed seedlings of three oak species for 4.5 months in an open air facility, using differing ozone (O3) and drought treatments: O3 (three levels from ambient to ×1.4 ambient), and drought (three levels of irrigation from 40% to 100% field capacity). We related the accumulated phytotoxic O3 dose (POD1) and cumulative drought index (CDI) to the O3 and drought VFI and assessed growth increment (height, diameter, leaf number), biomass (of all organs), and physiological parameters: net photosynthesis per plant (Pn), photosynthetic nitrogen (PNUE) and phosphorus use efficiency (PPUE)). The results indicated that an increase in POD1 promoted O3 VFI in Quercus robur and Quercus pubescens, while Quercus ilex was asymptomatic. The POD1-based critical level at the onset of O3 VFI was lower for Q. robur than for Q. pubescens (12.2 vs. 15.6 mmol m−2 POD1). Interestingly, drought reduced O3 VFI in Q. robur but increased it in Q. pubescens. Both O3 and drought were detrimental to the plant biomass. However, Q. robur and Q. pubescens invested more in shoots than in roots, while Q. ilex invested more in roots, which might be related to a hormetic mechanism. Pn, PNUE and PPUE decreased in all species under drought, and only in the sensitive Q. robur (PPUE) and Q. pubescens (PNUE) under O3. This study confirms that POD1 is a good indicator to explain the development of O3 VFI and helps a differential diagnosis of co-occurring drought and O3 VFI in oak forests.


Introduction
Tropospheric ozone (O 3 ) is an oxidative pollutant harmful to plants [1]. Ozone enters the leaves through the stomata, reacts in the mesophyll, and triggers the formation of reactive oxidative species (ROS) with a cascade of events eventually promoting cell death and, finally, the appearance of visible foliar injury (VFI), physiological impairment, and growth reduction [2][3][4]. Furthermore, O 3 inhibits the efficient use of nutrients such as nitrogen (N) and phosphorus (P) and thereby causes a reduction of photosynthetic N and P use efficiency (PNUE and PPUE, respectively) [5,6]. Therefore, critical levels (CL) have been investigated to assess the O 3 negative impacts on several plant species, especially those related to biomass loss [7,8]. CLs are based on cumulative O 3 indexes, e.g., AOT40, defined as the accumulated exposure over 40 ppb hourly concentrations, and PODy, defined as the phytotoxic O 3 dose above an hourly threshold y of stomatal O 3 uptake [9]. PODy is considered the most realistic index with a high correlation with the detrimental effects of O 3 [10,11]. Ozone VFI is a forest-health indicator in forest monitoring programs [12].
The details of the FACE facility are described in Paoletti et al. [26], and the details of the experimental design are published in Hoshika et al. [27].

Evaluation of O 3 and Drought Visible Foliar Injury
Two well-trained observers evaluated the presence of O 3 and drought VFI during the experimental period for all plants for a total of 6 evaluation dates (Table 1). We applied photo guides to verify whether O 3 and/or drought VFI was present [28][29][30]. VFI incidence (INC = number of injured plants/total number of plants × 100) was calculated according to Chappelka et al. [31]. POD 1 -based CLs and CDI-based CLs were calculated for the corresponding day when O 3 and drought VFI onset was observed.

Measure of Growth Parameters
The assessment of total annual biomass production during the experiment was performed based on dry weight per plant (DW) as described in Hoshika et al. [22], additionally discriminating the below-(roots) and above-ground biomass (stem and leaves) to calculate the ratio of root to shoot biomass (Ratio R/S). Furthermore, the total number of leaves, plant height increment (measured with a metric tape) and stem caliber increment (measured just above soil level) were expressed as the absolute values relative to the values at the beginning and end of the experiment.

Assessment of Photosynthetic Parameters
The net photosynthetic rate (P n ) was previously reported for mid-summer (July: [24]) and early and late summer and autumn (June, August, and October: [23]). Here, these published data of P n were re-analyzed to address the cumulative effects of O 3 and drought on the photosynthetic activity. The target leaves were fully sun-exposed leaves (4-6th from the shoot tip) of the plant main shoot (one representative leaf per plant, 1 to 3 plants per replicated plot per each O 3 and W treatment). Measurements were made under lightsaturated conditions (1500 µmol m −2 s −1 PPFD [photosynthetic photon flux density]) with constant CO 2 concentration (400 µmol mol −1 ), relative humidity (40 to 50%), and leaf temperature (25 • C) using a commercial gas exchange system (CIRAS-2 PP Systems, Herts, UK). Measurements were carried out in two campaigns (8-10 June and 27 September-6 October) for all O 3 treatments and an additional campaign (6-9 August) for two O 3 levels (1.2, and ×1.4) on days with clear sky between 9:00 and 12:00 a.m. CET. The other detailed specifications for the photosynthetic measurements were described in our previous studies [23,24].
After the measurement of P n in August and October, leaves were collected to examine the nitrogen (N) content. Nitrogen content per unit mass (N mass ) was determined by the dry combustion method using a LECO TruSpec C/N analyzer (Leco Corporation, St. Joseph, MI, USA). In October, the foliar phosphorus (P) content was also determined. Phosphorus content per unit mass (P mass ) was examined by an inductively coupled plasma-optical emission spectroscopy (ICP-OES) (iCAP7000, Thermo Fisher Scientific, Waltham, MA, USA). We calculated photosynthetic N use efficiency (PNUE) as the product of N mass and mass-based net photosynthetic rate and photosynthetic P use efficiency (PPUE) as the product of P mass and mass-based net photosynthetic rate.

Calculation of Accumulated Drought and Ozone Indexes
The accumulated drought index (CDI) was calculated from the beginning of the experimental period to the date of observation as follows: where, Sm is soil moisture, and Fc is field capacity (0.295 m 3 m −3 ) [26]; drought stress is considered severe when Sm values are lower than Fc.
AOT40 and POD 1 for each O 3 and drought treatment were calculated following the parameters applied by Hoshika et al. [22] according to the methodology designed by CLRTAP (Convention on Long-range Transboundary Air Pollution) [9].

Statistical Analysis
Multiple Linear Regression (MLR) analysis was used to estimate the relationship between the O 3 indexes (AOT40 and POD 1 ) and CDI versus growth (height, diameter, and N. leaves), biomass (Leaf, Shoot, Root, Total, and R/S), VFI (O 3 and Drought) and physiological parameters (P n , PNUE, and PPUE). Two models were compared, i.e., Model 1 (POD 1 and CDI as predictor variables) and Model 2 (AOT40 and CDI as predictor variables). The statistical analyses were performed using the R software (R version 4.1.2 [32]), considering a significance of p < 0.05. Principal component analysis (PCA) was conducted by using OriginPro 2021b software. The PCA was applied considering VFI (O 3 and drought), growth (Height, Diameter, and N. of leaves), biomass (Leaf, Shoot, Root; Total and R/S), and physiological (Pn, PNUE, and PPUE) parameters in order to distinguish the groups of parameters better related to each symptomatic species; in this analysis, the asymptomatic Q. ilex species was not included.

Visible Foliar Injury
The O 3 VFI in Q. robur was characterized by small homogeneously distributed dots between the primary leaf veins ( Figure 1A). Q. robur plants from all water regimes but SD (AA and ×1.2) presented O 3 VFI (Tables 1 and 2). In fact, 11% of the SD-treated plants developed O 3 VFI at the end of the experiment, relative to 56% of the WW-treated plants ( Table 2).
There were individual-specific differences on the day of VFI onset.  Table 1). In addition, the MLR revealed a positive regression of O 3 VFI with POD 1 or AOT40 and a negative regression with CDI when tested with AOT40 (Model 2), but the effect was not significant when tested with POD 1 (Model 1) ( Figure 1B; Table 3).    The O 3 VFI in Q. pubescens was similar to that developed by Q. robur ( Figure 1C). Independently of the water regime, plants from AA did not show O 3 VFI, while plants from ×1.2 and ×1.4 treatments presented O 3 VFI (Tables 1 and 2). The percentage of plants presenting VFI was lower than for Q. robur, with a maximum of 33% presenting VFI (Table 2). VFI occurred for the first time at DOY 247 or 266, i.e., around the end of the experiment. The POD 1 and AOT40 values for the O 3 VFI onset were 12.8-20.46 mmol m −2 POD 1 (average = 16.8 mmol m −2 POD 1 ) and 24-33 ppm h AOT40, respectively ( Table 1). The MLR revealed a positive regression with the O 3 indexes (POD 1 and AOT40; Figure 1D, Table 3). Interestingly, CDI positively affected the O 3 VFI when tested with POD 1 (Model 1), but the effect was not significant when tested with AOT40 (Model 2) ( Table 3).
The drought VFI of Q. robur was evident exclusively on the leaf edge that became dry and brownish (Figure 2A). The VFI progressively increased in MD-and SD-treated plants until the end of the experimental period, while WW-treated plants did not show any injury (Table 2). At the end of the experiment, 89-100% of the SD-treated plants showed drought VFI, relative to 63-67% of the MD-treated plants ( Table 2). The CDI calculated at the drought VFI onset was the same for all SD-treated plants (CDI = 10.20 for all AA, ×1.2, ×1.4 treatments, Table 1) and similar for MD-treated plants (CDI= 4.96 to 6.10, Table 1). The MLR revealed a strong positive regression between the Q. robur drought VFI and CDI, although POD 1 or AOT40 also increased the extent of drought VFI ( Figure 2B, Table 3). The drought VFI of Q. pubescens was similar to that developed by Q. robur ( Figure 2C). At the end of the experimental period, Q. pubescens presented 44-78% of the SD-treated plants with VFI, 11-33% of the MD-treated plants, and no VFI for the WW-treated plants. As found in Q. robur, the CDI calculated at the drought VFI onset was the same for all SDtreated plants (CDI = 10.20 for all AA, ×1.2, ×1.4 treatments) and similar for MD-treated plants (CDI= 4.04 to 6.52, Table 1). Interestingly, the CDI values at drought VFI onset were similar in Q. robur and Q. pubescens within the same O 3 and drought treatments (Table 1). In addition, the MLR revealed a positive regression with CDI, POD 1, and AOT40 (Table 3, Figure 2D). The evergreen Q. ilex did not present O 3 or drought VFI.

Physiological Responses
In both Q. robur and Q. pubescens, P n was negatively affected by POD 1 and CDI (Table 3, Figure S1B and D), but it unexpectedly increased with increasing AOT40 (Table 3). Furthermore, PNUE and PPUE were negatively related to CDI and POD 1 except for PNUE in Q. robur (Table 3).
For Q. ilex, the MLR revealed that CDI negatively affected P n ( Figure S1F), PNUE, and PPUE, with no significant relationship with the O 3 indexes (POD 1 and AOT40, Table 3).

Growth and Biomass
The MLR indicated that height increment was positively affected by POD 1 or AOT40 in Q. robur, while increments of diameter and number of leaves were negatively affected by CDI ( Table 3). As confirmed by negative regression coefficients with CDI, most biomass parameters of Q. robur were reduced by drought. On the other hand, POD 1 or AOT40 positively affected leaf biomass and negatively affected root biomass, indicating a reduction of the R/S ratio under elevated O 3 exposure (Table 3, Figure S1A).
In Q. pubescens, the O 3 indexes (POD 1 and AOT40) were positively related to plant height increment, while CDI was negatively related to height only when tested with AOT40 (Table 3). Increments in shoot diameter and number of leaves in this species were negatively related to POD 1 and AOT40, while they were negatively related to CDI when tested with POD 1 (Model 1, Table 3). Regarding the biomass parameters, leaf biomass was not affected by any factor, while shoot biomass was negatively affected by both O 3 indexes (POD 1 and AOT40) and CDI (Table 3). Root and total biomass were negatively related to CDI, and the R/S ratio was negatively influenced by CDI and POD 1 (Table 3, Figure S1C).
In Q. ilex, plant height increment was not affected by any factors, while a positive relationship between diameter increments and O 3 indexes was found ( Table 3). The increment in the number of leaves was positively affected by POD 1 and AOT40 and positively affected by CDI when tested together with POD 1 (Table 3), although leaf and total biomass were not significantly affected by those factors. Shoot biomass was negatively affected by CDI only when tested with AOT40, and root biomass was positively affected only by POD 1 ( Table 1). The R/S ratio was positively related only to POD 1 ( Table 3, Figure S1E).
The raw data off all growth parameters for the species Q. robur, Q. pubescens, and Q. ilex are avaible in Table S1.

Principal Component Analysis
The PCA detected separate multivariate spaces between the two symptomatic species as groups related to different growth, biomass, and physiological parameters related to O 3 or drought VFI (Figure 3). Since Q. ilex did not show VFI, this species was not included in the analysis. The first two components of the PCA explained 45.57 and 27.05% of the variances. The SD-treated plants of both species were grouped near the drought VFI (DS) with no other parameter following the same vector direction. The individuals of Q. robur (especially MD-treated plants) were grouped near the vectors of the growth parameters number of leaves and height, leaf biomass, and O 3 VFI, which presented the same direction, thus indicating that when O 3 VFI increased, these parameters also increased. The individuals of Q. pubescens (specially WW-treated plants) were grouped near P n , PNUE, and R/S, with the vectors in the opposite direction of O 3 and drought VFI, thus indicating that when O 3 and drought VFI increased, these parameters decreased.

Development of Visible Injury Due to Ozone and Drought Stress
The POD 1 values corresponding to the onset of O 3 VFI for the two symptomatic deciduous oaks (on average, 14.4 mmol m −2 ) were similar to those estimated for broadleaf species under field conditions in Italy and France (10 mmol m −2 s −1 ) [10]. However, the CL for the VFI onset was lower for Q. robur than for Q. pubescens, indicating its higher sensitivity to O 3 , possibly related to its lower antioxidative capacity and inability to protect the cell structure [25]. Furthermore, O 3 VFI increased with increasing POD 1 in the two deciduous oaks. This suggests that PODy is a key indicator to describe the development of O 3 VFI [33] once it is well known that O 3 damage is closely related to stomatal O 3 uptake [1]. In fact, the absence of O 3 VFI in Q. ilex might be related to its low g max (165 mmol O 3 m −2 s −1 , compared to 225 mmol O 3 m −2 s −1 and 200 mmol O 3 m −2 s −1 of Q. pubescens and Q. robur, respectively, [22] suggesting that the development of VFI might be discussed in terms of the specific-species patterns of stomatal conductance. For both injured species (Q. robur and Q. pubescens) at the end of the experimental period, the severe drought treatment reduced POD 1 by 30 to 40% [22], which would be expected to decrease the O 3 -induced VFI in plants as reported before for ecophysiological responses in poplars [34]. In Q. robur, the presence of O 3 VFI was decreased under drought. On the contrary, drought stress aggravated the O 3 VFI in Q. pubescens. Drought has been reported to have the potential to aggravate the harmful effects of O 3 [35]. Furthermore, Hoshika et al. [24] found that the combination of O 3 and drought altered the activity of the antioxidant system so that Q. pubescens was not protected from the severe oxidative stress resulting from the combined stress of O 3 and drought.
For the symptomatic species (Q. robur and Q. pubescens), the progression of drought VFI could be attributed to the obstruction of conducting tissue [20], conferring to both species a high sensitivity. In the asymptomatic Q. ilex, this phenomenon might not happen due to its capacity to increase the cell wall thickness by reinforcing the strength and rigidity of the secondary cell walls with hemicellulose and lignin deposition (data not published). Changes in lignin might function as physical desiccation tolerance and maintenance of protein integrity in drought-tolerant species [36], thus helping the photosynthetic recovery activity after re-watering from severe drought episodes [37]. The CDI threshold for the appearance of drought VFI in the two symptomatic species was higher in SD-treated than MD-treated plants, possibly due to the interaction with leaf aging, which is an important physiological and biochemical defense factor against drought stress [38]. In fact, most plants showed drought VFI in mid-or late-summer in both SD-treated and MD-treated plants when leaves were relatively old.

Effects of Ozone and Drought Stress on Growth and Biomass Parameters
For both deciduous species (Q. robur and Q. pubescens), height increment was higher when exposed to O 3 treatment. This phenomenon was verified in other species, such as Populus sp. [39], and it is possibly related to promoting a new leaf development as a compensative response against O 3 damage. However, the decrease in the number of leaves was eventually found to be due to O 3 exposure, which might be related to the potential O 3 phytotoxicity that triggers programmed cell death, promoting an increase in leaf senescence [40]. When combined with drought stress, the effect can be more substantial once the lack of water and nutrients promotes a decrease in new leaf development.
Both O 3 and drought stresses were detrimental to the plant biomass increment in all the oak species. In fact, the reduction of biomass due to drought stress is reported for many species and is related to the reduction of water content, diminished leaf water potential and turgor loss, promotion of stomatal closure, and decreased cell enlargement growth [41,42]. As previously revealed by Alonso et al. [21], drought stress does not protect holm oak from O 3 effects when considering the whole plant response. However, differences between the species responses must be considered when comparing the species sensitivity. For example, we observed that Q. robur and Q. pubescens invested more in shoots than in roots when exposed to both stresses, while Q. ilex performed the opposite, which might be another strategy of Q. ilex indicating a hormetic mechanism of tolerance for increasing conducting tissue and maintaining the water flow. These tolerance mechanisms may be associated with morphological/anatomical adjustments, such as a versatile root system, conservative growth and carbon allocation patterns, and diverse adaptations in the leaf morphology [20,43]. This might increase the apoplastic water fraction [44] and promote the species tolerance to O 3 and drought stress.

Effects of Ozone and Drought Stress on Physiological Parameters
The O 3 and drought stress negatively affected the physiological parameters. Drought stress induced a decrease of P n regardless of the different species, as confirmed by a negative relationship with CDI. A decrease in P n with increasing POD 1 was verified for both sensitive species (Q. robur and Q. pubescens), while no such reduction was found in Q. ilex. The present discussion is based on the species responses to POD 1 once the flux-based index is more realistic [9]. In fact, AOT40 was positively related to P n in the two deciduous oaks, which does not agree with a consensus about O 3 negatively affecting photosynthetic capacity [45]. In fact, the regression coefficient was very low (=0.000), although the regression slope was numerically significant. Even though data was generated from an underlying distribution, the significance is a rather unlikely biological sense. The data suggest that a biological-sound index such as POD 1 is superior to AOT40 for the studies of the O 3 effects on vegetations because it can consider the principal physiological cause of O 3 damage, i.e., stomatal O 3 uptake.
Drought stress decreased PNUE and PPUE for all three species, while O 3 stress negatively affected PNUE for Q. pubescens and PPUE for the sensitive species Q. robur and Q. pubescens. Drought stress is directly related to changes in the allocation of N and P to leaves, no matter the species sensitivity to O 3 stress. However, a reduced allocation of N and P to the photosynthetic apparatus [5,6,46] is more pronounced in O 3 sensitive species. The N-uptake efficiency and leaf N efficiency are important traits to improve growth under drought [47]; thus, the decline in root biomass might explain the decrease in PNUE and PPUE for those species, once reduced quantity of absorptive roots reduces water and nutrient uptake as verified for the same oak species in a previous study [48].

Is the Ozone Visible Injury an Indicator to Explain Biomass Reduction or Physiological Damage in Mediterranean Oaks?
The PCA biplot contains the strength of VFI, physiology, and growth relationships, along with the species-specific sensitivity to drought and O 3 stress. Relationships between O 3 VFI and biomass growth were discussed by other authors [49,50]. In the present study, we observed that the vector of O 3 VFI injury (O 3 S) and total biomass (TB) were crossing at the right angle of each other, suggesting a weak association between these two parameters in Mediterranean oaks. However, the O 3 S vector shows the same direction as those of leaf parameters (number of leaves [N.L] and leaf biomass [LB]) in plants presenting more O 3 VFI, which may indicate the promotion of carbon allocation to leaves as a compensation response against O 3 injury. In addition, opposite directions of the vectors were found for O 3 VFI (O 3 S) and net photosynthesis (P n ), PNUE, and the R/S ratio, highlighting a negative correlation between O 3 VFI and these parameters. The results indicate that O 3 VFI was not a direct indicator of biomass reduction under elevated O 3 in these oaks but provides important insights regarding the impairment of photosynthetic capacity and biomass partitioning to roots. Mediterranean oak species generally develop taproots that grow deep into the soil, enhancing resistance to abiotic stress such as drought [51]. However, small amounts of roots due to O 3 exposure imply a loss of water and nutrient uptake, suggesting that O 3 VFI should be considered a bioindicator in forests exposed to the combination of O 3 pollution and drought.

Conclusions
We examined O 3 -and drought-induced VFI and their effects on growth, biomass, and physiological parameters by using cumulative indexes and oak species known for showing differential sensitivity to these stressors. The increase in POD 1 promoted the development of specific O 3 VFI in the isohydric Q. robur and the intermediate Q. pubescens, while the anisohydric Q. ilex was asymptomatic. In Q. robur, the presence of O 3 VFI was decreased under drought probably because drought-induced stomatal closure reduced O 3 uptake and thus limited O 3 damage. However, drought stress aggravated O 3 VFI in Q. pubescens. This result indicates the importance of the protective role of antioxidant activity under the combination of O 3 and drought, which may be weakened by the combined stress factors and become a dominant factor in species that are not strictly isohydric. On the other hand, the drought VFI was clearly distinguished from the O 3 -induced VFI, and it developed with increasing CDI in Q. robur and Q. pubescens but not in Q. ilex, suggesting a high tolerance of Q. ilex to drought stress. Therefore, we suggest using the specific O 3 or drought VFI as a bioindicator, especially for establishing the onset injury CL.
We also confirmed that P n was decreased progressively with POD 1 and CDI in the two deciduous oaks, in tandem with PNUE decline, suggesting a cumulative effect of O 3 and drought on photosynthetic capacity. As a result, both stress factors showed a deleterious effect on the development of VFI and biomass growth. Interestingly, the two deciduous oaks increased the allocation to shoot growth rather than to root growth when exposed to both stresses, while an opposite result was found in Q. ilex. The imbalance in carbon allocation to roots may reduce the stability against strong winds and impair water uptake under the warming climate expected in future climate change [52,53].
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/plants11141836/s1, Figure S1: Results of the multiple linear regression for shoot/root (Ratio R/S) and Photosynthesis (P n ) parameters; Table S1: Raw data of growth parameters.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to technical limitations and other restrictions.