Ozone Flux-Based Response Functions for Visible Foliar Injury and Photosynthetic Traits in a Bioindicator Species, Viburnum lantana L.

Abstract

Tropospheric ozone (O₃) is a phytotoxic air pollutant that can impair visible foliar injury (O₃ VFI) and reduce photosynthesis in sensitive forest species. Viburnum lantana L. has been used as an in situ bioindicator of O₃ pollution in mountainous areas of Europe; however, flux-based response functions and critical levels (CLs) for this species have not yet been established. This study validated field-observed O₃ effects in V. lantana through experiments carried out in a Free-air O₃ eXposure infrastructure and determined which O₃ metric (exposure-based AOT40 or flux-based-POD₁) best explains O₃ effects on leaf physiology and VFI. Throughout the experimental period (T2: 3.5-month O₃ exposure), V. lantana saplings were subjected to ambient air (AA) conditions and elevated O₃ levels (1.5× and 2.0× AA). O₃ VFI appeared after 16 days in 2.0× and increased progressively during the growing season, reaching the highest Plant Injury Index (PII) values in the 2.0× (9.06 ± 3.24) compared with 1.5× and AA treatments (1.31 ± 0.62 and 1.29 ± 0.71). Elevated O₃ also significantly reduced net photosynthetic rate (A_sat), relative chlorophyll content (SPAD), and the maximum photochemical efficiency of photosystem II (F_v/F_m); no significant difference in stomatal conductance (g_s) was found. The flux-based metric POD₁ better explained variability in O₃ VFI and physiological parameters. Based on the best-fitting models, CLs for V. lantana were estimated at 1.61 mmol m⁻² and 1.22 mmol m⁻² for a 4% reduction in A_sat and g_s, and a CL of 7.82 mmol m⁻² for the O₃ VFI-onset.

1. Introduction

Tropospheric ozone (O₃) is an oxidative atmospheric pollutant that negatively affects forest health. Despite regulatory efforts to reduce precursor emissions, O₃ concentrations across much of Europe continue to exceed the thresholds established for protecting ecosystems [1].

Upon entry into the leaves via the stomata, O₃ interacts with the mesophyll, triggering production of reactive oxygen species (ROS), which subsequently cause physiological impairment, visible foliar injury (O₃ VFI), and finally growth loss [2,3], while leaf phenotypic plasticity modulates plant sensitivity [4]. To investigate tree responses to O₃, several metrics have been developed, including the exposure-based index Accumulated Exposure Over a Threshold of 40 ppb (AOT40) [5]. However, flux-based indices, such as the phytotoxic O₃ dose above a flux threshold of Y nmol m⁻² s⁻¹ (POD_Y), have been proven to be more reliable and account for the actual O₃ dose determined by cumulative O₃ uptake over the growing season [6].

O₃ VFI serves as a forest-health biomarker within monitoring initiatives, e.g., the International Co-operative Program on assessment and monitoring of air pollution effects on Forests (ICP Forests) [7]. This is because the O₃ VFI assessment is practical for evaluating O₃ impact on forests in the field without specialized equipment, making it suitable for long-term monitoring. Therefore, scientific efforts have been made to establish O₃ Critical Levels (CLs) for forest protection based on the assessment of O₃ VFI for some dominant tree species (e.g., Fagus sylvatica L., Pinus halepensis Mill., Picea abies L. Karst.) [8,9] and other forest tree or shrub species (e.g., Betula pendula Roth, Larix decidua Mill., Populus tremula L., Salix caprea L., Rubus sp., Vaccinium myrtillus L.) [10,11]. However, validation of O₃ VFI using manipulative experiments is still required to establish an appropriate CL for forest protection [12].

In recent decades, Viburnum lantana L. has been proposed as an in situ bioindicator of O₃ damage due to its foliar sensitivity and wide distribution in European forests. Field studies in the Italian Alps found “O₃-like” VFI (red stippling and leaf reddening), which was correlated with high O₃ levels (AOT40 > 30 ppm h), though symptom severity also reflected microclimatic conditions, indicating that the CL for O₃ VFI in this species is around 12 ppm h AOT40 [13]. Follow-up research confirmed these patterns, highlighting inter-annual variability likely linked to climate-driven differences in O₃ uptake [14,15]. Long-term monitoring through the ViburNeT network [14,15] further supported these findings, documenting a consistent relationship between regional O₃ concentrations and O₃ VFI frequency in V. lantana, with 20.7% to 50.6% of individuals exhibiting O₃ VFI frequency, depending on the year. More recently, Faralli et al. [16] demonstrated that symptom severity showed a significant association with lower specific leaf area (SLA) and higher trichome density, especially under high light exposure and in the upper canopy. These findings, although valuable, are based on field observations, where site-specific susceptibility of V. lantana to local environmental conditions can influence stomatal O₃ uptake and thus alter the response to O₃. To date, there has been no study evaluating O₃ damage to V. lantana on the basis of stomatal O₃ flux. Therefore, to fully understand and validate its biomonitoring potential for setting the biological O₃ standard, it is essential to investigate the species’ O₃ response under controlled conditions, such as through Free-Air Controlled Exposure (FACE) systems, aiming to establish a flux-based CL for this bioindicator species.

The present study enables assessments of O₃ VFI and physiological responses in the forest bioindicator species V. lantana, with the primary aim of reducing the influence of uncontrolled site-specific environmental variability and validating field-based observations under controlled conditions.

The V. lantana plants were exposed to different O₃ levels at the Free-Air O₃ eXposure (FO₃X) facility, with the specific objectives being: (i) to characterize the onset, progression, and severity of O₃ VFI in V. lantana under open-air experimental conditions; (ii) to assess the effects of O₃ exposure on key photosynthetic traits, including light-saturated net photosynthetic rate, stomatal conductance, relative chlorophyll content, and PSII photochemical efficiency; and (iii) to determine which indices between exposure-based (AOT40) and flux-based (POD₁) more accurately capture the effects and incidence of O₃-related VFI and photosynthetic impairment in the bioindicator species V. lantana. The experiment was designed to test whether increasing O₃ exposure induces a dose-dependent increase in VFI and a decline in photosynthetic performance in V. lantana. Because POD₁ accounts for the stomatal O₃ dose actually taken up by the leaves, this flux-based index was expected to explain O₃-induced responses more effectively than the exposure-based metric AOT40.

2. Materials and Methods

2.1. Experimental Site and Plant Material

Ozone exposure experiments were conducted at the FO₃X facility situated within the experimental garden of the National Research Council of Italy (Sesto Fiorentino, Italy: 43°48′59″ N, 11°12′01″ E, 55 m above sea level, Figure 1A,B). The fumigation system details are provided in Paoletti et al. [17] (close-up view of the free-air fumigation system Figure 1C). In December 2023, three-year-old Viburnum lantana L. saplings, raised in Santa Giustina in Colle, were obtained from a local nursery (Vivai Guagno, S. Giustina in Colle, Padova, Italy) with an average height of 74.7 cm and a stem diameter of 13.5 mm. Each plant was transplanted into a 9 L plastic pot (24 cm diameter) containing a uniform mixture of sand, peat, and soil in equal parts (v:v:v). This substrate composition was selected to ensure adequate drainage, nutrient availability, and root aeration during the subsequent growth period. All plants were watered daily to prevent water stress. From 17 May to 16 October 2024 (152 days), the plants were exposed to three levels of O₃ concentration treatments (ambient O₃ concentration [AA], 1.5 times ambient O₃ concentration [1.5×], and doubled ambient O₃ concentration [2.0×]). A total of 18 plants were used in the experiment, with two plants per plot and three plots (5 m × 5 m × 2 m) per treatment. The low number of plants per treatment was a limitation of the present study, reflecting an operational constraint of the facility during this experimental year, and was considered when interpreting the statistical outcomes.

In addition to continuous monitoring of O₃ concentrations (Mod. 202, 2B Technologies, Boulder, CO, USA), environmental variables such as air temperature, solar radiation, and precipitation were also continuously recorded (Watchdog station, Mod. 2000; Spectrum Technology, Inc., Aurora, IL, USA), and are reported in Figure 1D–F. The average hourly O₃ concentrations in the AA, 1.5×, and 2.0× treatments were 38.0 ppb, 48.6 ppb, and 61.3 ppb, respectively.

2.2. Assessment and Quantification of Visible Foliar Injury

Visible foliar injury attributable to O₃ was monitored at 2 or 3 week intervals throughout the exposure period. Assessments were carried out independently by two trained and calibrated observers. For each plant, we recorded the proportion of leaves showing symptoms (SL) and, within symptomatic leaves, the mean affected leaf area (SA), using a ×10 hand lens and published photographic references [18,19]. To quantify the injury at the plant level, the Plant Injury Index (PII) was calculated by combining the two parameters [20] as follows:

$$PII={\displaystyle \frac{\left(SL\times SA\right)}{100}}$$

(1)

To describe, classify, and standardize O₃ VFI, color composition was assessed based on the methodology suggested by Moura et al. [12]. Three pictures showing a range of injuries, visually divided into no O₃ VFI, low O₃ VFI, and high O₃ VFI, were taken under natural light conditions on the same day between 9:00 and 10:00 a.m. Images were captured using a high-resolution digital camera (Cyber-shot DSC-H300, Sony, Tokyo, Japan), with the white balance manually adjusted to ensure accurate color representation. The photos were processed in Adobe Photoshop 2026 (Adobe Inc., San Jose, CA, USA) and first combined into a single RGB figure. Subsequently, using the Indexed Color mode with Local Perceptual settings, the figure’s color palette was reduced to 64 colors. All the tissue exhibiting greenish and the vein-related colors were extracted from the image, leaving only potential O₃ VFI colors visible.

2.3. Assessment of Photosynthetic Parameters

Measurements of photosynthetic parameters (i.e., SPAD, chlorophyll a fluorescence, and leaf gas exchange) were conducted at three time points (time zero—T0: 13 May [before O₃ exposure], T1: 11 July [55 days exposure], and T2: 8 September [114 days exposure]). Although O₃ exposure continued until 16 October, T2 was selected as the final physiological measurement point to avoid the onset of leaf senescence, which could affect physiological responses.

Leaf gas exchange rates were measured using a portable photosynthesis measurement system (LI-6800, Li-Cor instruments, Lincoln, NE, USA). Measurements were carried out between 08:00 and 12:00 CET at each time point on all 18 exposed plants. For each plant, measurements were taken on leaves of the 4th to 6th order from the shoot tip. Across the three time points, a raw dataset of 108 leaf-level measurements was produced. For statistical analysis, leaf-level measurements were averaged within each Time × Plot × O₃ treatment combination, which was used as the final statistical unit. During the measurement, the parameters within the LI-6800 leaf cuvette were established as follows: CO₂ concentration (420 ppm), leaf temperature (25 °C), photosynthetic photon flux density (PPFD, 1500 μmol m⁻² s⁻¹, utilizing an LED light source of 10% blue and 90% red light), and relative humidity (50%). From these measurements, light-saturated net photosynthetic rate (A_sat) and stomatal conductance (g_s) were determined.

A SPAD meter (Konica Minolta, Tokyo, Japan) was utilized to measure leaf greenness as a proxy of chlorophyll content. In addition, a HandyPEA fluorimeter (Hansatech Instruments, Pentney, Norfolk, UK) was used to measure the chlorophyll a fluorescence. In order to measure the chlorophyll a fluorescence, after 40 min of dark adaptation, leaves were subjected to a 1 s saturating pulse of red light (peak wavelength: 650 nm) at an intensity of 3000 μmol photons m⁻² s⁻¹ to determine the fluorescence yields in the dark (F₀) and those with a saturating pulse (F_m). The maximum quantum yield (F_v/F_m) was calculated as F_v/F_m = 1 − (F₀/F_m).

2.4. Modeling of Stomatal Conductance

In order to estimate stomatal O₃ flux, it is essential to parameterize the g_s model. A measurement campaign of g_s for the 4th to 6th ordered leaves of all target plants was conducted using a porometer (LI-600, Lincoln, NE, USA) to ensure a large number of measurements. Stomatal conductance was measured on 19 sampling days, along the experimental period (from T0 to T2), in order to cover a range of environmental conditions. The resulting dataset, comprising 593 measurements, was used to parameterize the multiplicative g_s model [21,22], as described below:

$$g_s=g_{max}·f_{light}·max\left\{f_{min,} \left(f_{temp}·f_{VPD}\right) \right\}$$

(2)

where g_max is the maximum stomatal conductance (mol O₃ m⁻² Projected Leaf Area [PLA] s⁻¹). All other functions are expressed in relative terms and scaled from 0 to 1. The model incorporates minimum stomatal conductance (f_min), and it adjusts g_s based on PPFD (f_light), temperature (f_temp), and vapor pressure deficit (VPD) (f_VPD). The details of the model functions are available in the Supplementary File (Supplementary Method S1). In this study, the function of soil water content (f_SWC) was not included because plants did not receive any soil water stress. The model was parameterized using the boundary line approach [23] as a recognized technique to assess the impact of O₃ on plant productivity [24]. The g_max and f_min values were set as the 95th and 5th percentiles of all g_s data, respectively, following the methodology of Bičárová et al. [25].

2.5. Calculation of Ozone Indices

For daylight periods, identified by shortwave radiation exceeding 50 W m⁻², AOT40 was obtained by summing the hourly exceedance of O₃ concentrations above the 40 ppb threshold, following CLRTAP [26]. It is defined as:

$$AOT40={\sum }_{i=1}^{n}max\left({\left[O_3\right]}_i-40.0\right)$$

(3)

where [O₃]i represents the measured hourly O₃ concentration (ppb), with i ranging from 1 to n in the summation, where n is the total number of hours in the calculation period.

According to CLRTAP [26], stomatal O₃ flux (F_st; nmol m⁻² s⁻¹) can be given as:

$$F_{st}=\left[O_3\right]·g_s·{\displaystyle \frac{r_c}{r_b+r_c}}$$

(4)

where [O₃] (ppb) is the hourly O₃ concentration, r_c is the surface resistance of leaf (=1/(g_s + g_ext); s m⁻¹), g_s is the stomatal conductance (m s⁻¹), and r_b is the boundary layer resistance of the leaf (s m⁻¹) calculated as r_b = 1.3 ×150 × (L_d/u) ^0.5 where u is the wind speed (m s⁻¹), L_d is the cross-wind leaf dimension (0.05 m for broadleaves [26]).

POD_Y (mmol m⁻²) was calculated as the sum of hourly F_st data as:

$${POD}_Y={\sum }_{i=1}^{n}max\left(F_{st\_i}-Y, 0 \right)$$

(5)

where F_{st_i} is the hourly stomatal O₃ uptake (nmol m⁻² s⁻¹), n is the number of hours included in the calculation period. Y is a species-specific threshold of stomatal O₃ uptake (nmol m⁻² s⁻¹). In the mapping manual [26], Y = 1 nmol m⁻² s⁻¹ is recommended to assess the negative O₃ effects on woody plant species. Therefore, we utilized POD₁ to establish dose–response relationships with O₃ VFI and physiological parameters.

2.6. Data Analysis

To assess treatment effects, PII and physiological parameters (A_sat, g_s, SPAD, and F_v/F_m) were analyzed on absolute values by two-way ANOVA with O₃ treatment and time as fixed factors, including their interaction, after verifying normality and homogeneity of variances. When significant effects were detected (p ≤ 0.05), differences among levels were evaluated using Tukey’s HSD post hoc test.

Dose–response relationships were analyzed separately by expressing each response variable as a relative change from an operational baseline, defined by the mean value measured under AA at T0 (considered as the reference condition in which features were unaffected by O₃). Exposure- and flux-based dose–response functions were then fitted using AOT40 or POD₁ as predictors, comparing: (i) a linear model and (ii) a non-linear polynomial model (proposed for O₃ risk assessment applications by Hoshika et al. [27]). Model selection was performed using the Akaike Information Criterion (AIC), a widely adopted approach in ecological research that balances model fit with complexity by penalizing the inclusion of additional parameters [28]. This criterion was shown to be particularly suited for O₃ dose–response comparisons [29,30] and was used to identify the model with the best predictive accuracy. Among the candidate models evaluated, the one with the lowest AIC value was selected as the best-fitting model, reflecting an optimal trade-off between explanatory power and parsimony. The coefficient of determination (R²) was used to assess the proportion of variance explained by the selected model.

A sensitivity scenario analysis was also conducted to quantify how assumptions on stomatal regulation affect flux-based estimates. Specifically, when AOT40 reached 12 ppm h (as suggested by Gottardini et al. [13]), POD₁ was recalculated under a set of imposed stomatal-closure scenarios (0–50% closure, 10% increments) to simulate additional environmental stress (e.g., drought).

For VFI, CLs were defined as the exposure/uptake at which symptoms first occurred, operationally identified as the point at which PII became > 0 (PII = 0.01). For physiological parameters (A_sat, g_s, SPAD, and F_v/F_m), the CL was defined as the dose corresponding to a 4% reduction relative to the baseline, following Hoshika et al. [27]. All statistical analyses and graphical plotting were performed using OriginPro 2025 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Ozone Visible Foliar Injury and Plant Injury Index

The first O₃ VFI for the plants treated with 2.0× O₃ exposure were observed after 16 days of exposure (on 2 June). Subsequently, the symptoms emerged in the other two O₃ treatments (i.e., AA and 1.5×) 32 days after the beginning of the exposure (on 18 June). The O₃ VFI was characterized by interveinal stippling on the adaxial leaf surface, with a reddish to dark brown coloration (Figure 2). These stipples were localized between the veins, often sparing the vein tissue itself, and did not show signs of tissue necrosis or insect damage.

Color-composition analysis identified 16 perceptual colors that captured the main chromatic features of O₃ VFI in V. lantana (Figure 2). The selected palette described a gradual shift from light to dark brownish tones, with reddish intermediates, consistent with the visual progression of injury.

The PII was significantly affected by the interaction between treatment and time (p < 0.05,

Table 1. Plant Injury Index (PII) and physiological traits measured (light-saturated net photosynthetic rate [A_sat], stomatal conductance [g_s], SPAD, the maximum photochemical efficiency of PSII [F_v/F_m]) in Viburnum lantana plants under ambient air (AA), 1.5× AA, and 2.0× AA O₃ treatments. Measurements were taken before exposure (T0, 13 May), after 55 days (T1, 11 July) and after 114 days (T2, 8 September). Values are reported as plot means ± SE. Significance levels from two-way ANOVA are indicated as *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and – = not significant. Different Greek letters denote significant differences in O₃ × time interactions, capital letters indicate statistical differences among treatments, and lowercase letters indicate statistical differences among time points (Tukey’s HSD test).

O3 × Time		PII	Asat µmol m−2 s−1	gs µmol m−2 s−1	SPAD	Fv/Fm
T0	AA	0.00 ± 0.00 α	14.68 ± 1.03 Aa	0.20 ± 0.03 a	44.40 ± 1.15 A	0.75 ± 0.00 α
	1.5×	0.00 ± 0.00 α	13.08 ± 0.75 ABa	0.14 ± 0.01 a	43.77 ± 1.37 B	0.73 ± 0.01 α
	2.0×	0.00 ± 0.00 α	12.85 ± 0.91 Ba	0.17 ± 0.01 a	40.43 ± 3.17 AB	0.75 ± 0.01 α
T1	AA	0.44 ± 0.12 α	8.05 ± 0.22 Ab	0.07 ± 0.00 b	52.10 ± 0.64 A	0.73 ± 0.01 α
	1.5×	2.03 ± 1.11 α	7.47 ± 1.13 ABb	0.10 ± 0.01 b	39.90 ± 3.55 B	0.71 ± 0.02 α
	2.0×	2.12 ± 1.41 α	7.92 ± 1.02 Bb	0.10 ± 0.01 b	41.27 ± 5.78 AB	0.64 ± 0.01 β
T2	AA	1.29 ± 0.71 α	8.62 ± 0.32 Ab	0.09 ± 0.01 b	54.77 ± 2.54 A	0.74 ± 0.01 α
	1.5×	1.31 ± 0.62 α	6.68 ± 0.65 ABb	0.10 ± 0.01 b	42.97 ± 5.49 B	0.75 ± 0.02 α
	2.0×	9.06 ± 3.24 β	3.73 ± 0.60 Bb	0.08 ± 0.02 b	46.77 ± 5.20 AB	0.72 ± 0.01 α
ANOVA	O3	*	**	–	*	**
	Time	**	***	***	–	**
	O3 × Time	*	–	–	–	**

). Across all O₃ treatments, PII values increased over time, with O₃ VFI already evident but not statistically significant at T1. At this stage, mean PII values exceeded 1.5 in the two elevated O₃ treatments (2.03 ± 1.11 in 1.5× and 2.12 ± 1.41 in 2.0×), reaching maximum values of 0.61 in AA, 3.83 in 1.5×, and 4.79 in 2.0×. Injury became even more pronounced at T2, with a statistical difference under the 2.0× treatment. At T2, while the AA showed modest increases (mean values were 1.29 ± 0.71), the 1.5× O₃ treatment showed a decrease, justified by leaf senescence in this treatment (1.31 ± 0.62), and a significant rise in PII was observed in the 2.0× treatment at T2 (9.06 ± 3.24). Variability in PII was high across treatments; the lowest PII value was 0.01, recorded in the 1.5× at T1 (i.e., 1.00% of SL and 10% of SA), and the highest recorded value of 14.13 was in 2.0× T2 (i.e., 41.3% of SL and 34.21% of SA).

3.2. Ozone Effects on Photosynthetic Parameters

The A_sat was significantly affected by both treatment (p < 0.05) and time (p < 0.001) (Table 1). Across treatments, a consistent decline was observed from AA to 2.0×, with AA presenting an average A_sat of 10.45 ± 1.10 µmol m⁻² s⁻¹, 1.5× an intermediate value of 9.07 ± 1.09 µmol m⁻² s⁻¹, and 2.0× an average of 8.16 ± 1.38 µmol m⁻² s⁻¹. Across time, A_sat at T1 (7.81 ± 0.45 µmol m⁻² s⁻¹) and T2 (6.34 ± 0.76 µmol m⁻² s⁻¹) were statistically lower than the baseline (T0: 13.54 ± 0.53 µmol m⁻² s⁻¹).

Regarding g_s, there was only a statistically significant influence of time (p < 0.0001) (Table 1), with mean values decreased from 0.172 ± 0.014 µmol m⁻² s⁻¹ at T0 to 0.091 ± 0.007 mol m⁻² s⁻¹ at T1 and remaining at a similar level at T2 (0.091 ± 0.006 µmol m⁻² s⁻¹).

SPAD values, indicative of relative chlorophyll content, were significantly affected by the O₃ treatment (p < 0.05, Table 1). Across all time points, the AA plants showed the highest mean SPAD values (50.42 ± 1.76), which were significantly higher than those observed in the 1.5× treatment (42.22 ± 2.12). In contrast, the 2.0× treatment group exhibited intermediate SPAD values (42.82 ± 6.62), which did not differ significantly from either AA or 1.5× due to the high within-group variability.

F_v/F_m was significantly affected by the interaction between treatment and time (p < 0.01; Table 1). Across all three O₃ treatments, F_v/F_m values remained stable throughout the experiment, with mean values of 0.72 ± 0.01, indicating limited temporal variation in PSII photochemical efficiency. A notable exception, however, was observed in the 2.0× treatment, which showed a significant decrease in F_v/F_m during mid-summer (T1: 0.64 ± 0.01), followed by a recovery at T2. This transient reduction was not observed at the other two exposure levels, which maintained consistent F_v/F_m values across all time points.

3.3. Parameterization of the Stomatal Conductance Model

The g_max value found for V. lantana was 0.158 mol O₃ m⁻² PLA s⁻¹, whereas f_min was set to 0.06. The limitations of g_s due to environmental factors are shown in Figure 3. With increasing light intensity, a sharp increase in g_s was observed, even under a relatively low PPFD (200 μmol m⁻² s⁻¹), as shown by f_light (f_{light_a} = 0.0163). The optimal temperature for stomatal opening (T_opt) was 24 °C, while T_max and T_min were set to 41 and 0 °C, respectively. In addition, a high air VPD observed in the afternoon caused stomatal closure, as confirmed by the f_VPD function (VPD_max: 1.8 kPa, VPD_min: 6.4 kPa).

3.4. The Relation of O₃ Indices, Plant Injury Index (PII), and Plant Physiological Responses

The results of the linear and polynomial regression models examining the relationships between PII and the physiological parameters (A_sat, g_s, SPAD, and F_v/F_m) and the two indices (POD₁ and AOT40) showed varying levels of model fit and explanatory power (Table S1). All the regressions were statistically significant except for SPAD and F_v/F_m, and thus will not be discussed in the study.

For the PII, the polynomial model performed better than the linear one, and the AOT40 (Figure 4A) performed slightly better than POD₁ (Figure 4B). As it was clear that the PII response remained flat across time of exposure and then sharply rose over a narrow interval at the end of the exposure period, we considered both polynomial models (with AOT40 and POD₁) as suitable for representing the species response (Figure 4A,B).

The best-fit model for physiological parameters was the polynomial, performed with POD₁ (Figure 4C,D). The A_sat declined rapidly at low POD₁, then approached a stable lower plateau at higher POD₁. However, A_sat remained well below its initial level across the higher POD₁ values, especially at the end of the exposure period, indicating reduced leaf photosynthetic capacity with increasing accumulated O₃ uptake (Figure 4C).

For g_s, the polynomial function using POD₁ also provided the best fit, showing a lower AIC compared to all the other tested functions (Table S1). The fitted curve was similar to that for A_sat, with g_s decreasing sharply at low POD₁, then stabilizing, when g_s had already declined substantially from its initial levels, and continued to decrease gradually as POD₁ increased (Figure 4D).

3.5. Critical Levels

The exposure- or flux-based CLs were calculated for all significant regressions (Table S1), and

Table 2. Ozone critical levels (CLs) for visible foliar injury and physiological responses in Viburnum lantana were estimated through the Plant Injury Index (PII), light-saturated net photosynthetic rate (A_sat), and stomatal conductance (g_s). Two metrics of ozone exposure were examined and tested using the polynomial model: the stomatal flux-based Phytotoxic Ozone Dose above a threshold of 1 nmol m⁻² s⁻¹ (POD₁), and the exposure-based AOT40 (Accumulated Ozone Exposure Over a Threshold of 40 ppb). The CLs for PII were calculated considering the injury onset (PII = 0.01), and for the photosynthetic parameters were derived as 4% reduction from the baseline value.

Variable/O3 Index	AOT40 (ppm h)	POD1 (mmol m−2)
PII	4.42	7.82
Asat	2.27	1.61
gs	1.31	1.22

reports only the CLs of the chosen best-fit functions.

Comparing the CLs calculated for the PII, the values were considerably higher with the polynomial function (7.82 mmol m⁻² for POD₁ and 4.42 ppm h for AOT40, Table 2) than with the linear regressions (0.54 mmol m⁻² for POD₁ and 0.81 ppm h for AOT40; Table S1).

In contrast, for the physiological parameters, the polynomial fits produced lower CLs than the linear fits, and g_s consistently showed lower CLs than A_sat, indicating an earlier response of stomatal conductance to O₃ (POD₁: 1.22 vs. 1.61 mmol m⁻²; AOT40: 1.31 vs. 2.27 ppm h, respectively; Table 2).

4. Discussion

The manipulative experimental approach adopted in our study for the first time enabled the following new findings on O₃ effect on both O₃ VFI and the physiological features of the important bioindicator species, V. lantana, filling a significant knowledge gap regarding the integrated response of the species to O₃ stress.

4.1. Validation of Ozone Visible Foliar Injury Based on Free-Air Experiments

The appearance and progression of O₃ VFI observed in V. lantana under elevated O₃ treatments confirmed that this species is sensitive to O₃ exposure. Plants exposed to 2.0× developed VFI two weeks earlier than those at lower treatment levels, following a clear dose-dependent pattern. This observation aligns with previous experimental studies on O₃ VFI for this species, which found that higher AOT40 levels were associated with more frequent VFI [14]. Previous studies suggest that the onset timing of O₃ VFI shows species-specific dependency; for instance, the O₃-sensitive deciduous species, Alnus glutinosa and Vaccinium myrtillus, showed early-season symptom development, whereas the O₃-tolerant Mediterranean shrub, Arbutus unedo, exhibited a delayed onset of O₃ VFI, appearing only in the late growing season [12]. In our study, although the O₃ VFI onset was found in the early summer, PII values at 2.0× treatment became significantly higher than in the AA treatment only at T2, likely reflecting the slow development of O₃ damage in V. lantana throughout the growing season.

The symptoms consisted of small reddish-brown spots distributed on the upper leaf surface, mainly in the interveinal areas. Veins were generally unaffected, and the leaves did not show necrotic patches or damage patterns attributable to insects or pathogens [7,18]. The color analysis (Figure 2) supported this pattern, showing a chromatic gradient from light gray to dark brown, indicative of increasing tissue alteration. Similar color changes have been described in previous field assessments of O₃ VFI in V. lantana [13], and with the general symptomatology reported for O₃-sensitive native species [11]. These colorimetric patterns not only reflect the severity of foliar damage but may also serve as a diagnostic tool for O₃ injury under field conditions. As demonstrated by Moura et al. [12], the color components observed in experimental manipulations are comparable in many species to those detected in field samples, confirming the comparability between controlled and natural environments. This suggests that the color profiles documented in our study could be effectively used for quality control and support field-based O₃ VFI assessments, enhancing the accuracy of visual diagnosis in biomonitoring applications.

Despite the clear development of O₃ VFI, the correspondence with systemic eco-physiological responses was only partial, highlighting the complexity of O₃–plant interactions. While PII increased significantly with O₃ exposure and A_sat showed a marked decline, the response of g_s was more limited. The decrease in A_sat in the absence of g_s reduction in O₃-exposed leaves was similarly found in Japanese Siebold’s beech [31]. More broadly, meta-analytic evidence indicates that O₃ adversely impacts both photosynthesis and stomatal conductance in trees, although the extent of these responses may vary among species and experimental conditions [32]. Since the rates of damage to photosynthesis and stomatal conductance do not always coincide, O₃ exposure can decouple g_s from A_sat [33]. In fact, O₃ impairs stomatal function, leading to reduced stomatal responsiveness to environmental stimuli, i.e., stomatal sluggishness [3,33,34]. The remaining open stomata reduce water-use efficiency, increasing the risk of other climate crisis factors, such as drought [32,35].

In our study, this partial decoupling between O₃ VFI and physiological impairment may be linked to ‘invisible’ damage and physiological modifications, including repair processes, before visible signs of injury appear [36,37]. In fact, in our experiment, the marked reduction in A_sat was already found at T1, even though PII was still relatively low. Moreover, a significant decline in F_v/F_m was observed at 2.0× only at the early time point (T1), suggesting an initial impairment of PSII photochemical efficiency. The role of Rubisco and Rubisco activase in mediating the O₃ effect in declining carboxylation efficiency has been well explored [38], but in the case of V. lantana the absence of sustained effects at later stages of exposure may indicate a partial recovery over time or a localized, non-systemic damage response. Similar dynamics were reported by Bussotti et al. [39] in a field-based study on V. lantana, in which chlorophyll fluorescence measurements under high O₃ exposure showed a marked reduction in parameters related to electron transport efficiency.

This mismatch between early physiological impairment and later visible injury is particularly relevant for upscaling O₃ impact assessment, since recent studies using solar-induced chlorophyll fluorescence (SIF) have shown that functional indicators can detect O₃-related stress before visible symptoms become apparent. The TROPOSIF dataset developed by Guanter et al. [40] provides global SIF retrievals from TROPOMI/Sentinel-5P, offering daily observations with denser spatial and temporal sampling than earlier satellite missions. Building on this type of product, Mamić et al. [41] combined plant-level O₃ exposure experiments with multi-year TROPOMI/Sentinel-5P SIF observations and showed that satellite-derived fluorescence can support the detection of O₃-related vegetation stress across broader spatial scales. Nonetheless, they emphasized that leaf-level experimental data and satellite observations are not directly comparable because they pertain to different spatial scales. Future investigations could integrate ground-based O₃ VFI assessments, flux-based response functions, meteorological data, stomatal modeling, and satellite-derived fluorescence or atmospheric products to improve the spatial extrapolation of O₃ risk assessments.

Overall, these findings confirm the moderate physiological sensitivity of V. lantana to O₃, with photochemical alterations detectable even before O₃ VFI becomes widespread.

4.2. Flux-Based Assessment of Ozone Visible Injury and Physiological Parameters

This study also aimed to evaluate whether O₃-induced effects on V. lantana are better captured by the flux-based index (POD₁) rather than the exposure-based index (AOT40). The parameterization of the g_s model, crucial for calculating POD₁, highlighted that the detected g_max was similar to the value found for Vaccinium myrtillus (i.e., 0.140 mol O₃ m⁻² PLA s⁻¹), another widespread shrub in European forests, previously studied by Hoshika et al. [42]. Moreover, the relatively high value of the parameter f_{light_a} underlined a rapid opening of the stomata in response to light availability, indicating a shade-tolerant behavior for V. lantana.

Regression analyses confirmed that POD₁ was a superior metric for capturing the physiological response, with the polynomial model fitting better than the linear regression, specifically, for A_sat and g_s. This is consistent with the wider application of flux-based dose–response relationships for assessing ozone impacts on European forest species [43]. While both O₃ indices were significantly related to PII, the polynomial model with POD₁ again showed a tendency toward a better fit and better represented the biological process (Figure 4B, Table S1).

The stronger association between POD₁ can be attributed to its physiological basis. Unlike exposure-based metrics such as AOT40, which rely solely on ambient O₃ concentrations, POD₁ estimates the actual O₃ dose absorbed through stomata. This flux-based approach is widely recognized as the primary driver of O₃-induced phytotoxicity [3], as it accounts for plant-specific factors such as stomatal regulation under varying environmental conditions (e.g., water availability). Furthermore, we recommend using non-linear models for dose–response relationships, as emphasized by Hoshika et al. [27], particularly when they outperform linear regression.

4.3. Critical Levels in Viburnum lantana

The use of the PII offers an integrated measure of O₃ VFI damage by combining both the proportion of symptomatic leaves per plant (SL) and the average affected area within those leaves (SA), while the use of POD₁ as the reference metric is consistent with flux-based approaches for defining biologically relevant O₃ CLs [44]. This dual-component approach provides a more realistic representation of total O₃ VFI than frequency-based metrics alone. In our study, we proposed that the CL for PII should be calculated when the PII value reaches 0.01, indicating the onset of damage (i.e., 1% SL and 10% SA), which, in the case of V. lantana, using the polynomial regression as the best fit (Table S1), occurred when POD₁ reached 7.82 mmol m⁻² (Table 2).

If calculated using the exposure-based index AOT40, the polynomial regression fit better, and the CL for the PII was determined to be 4.42 ppm h. In the past, a threshold of 12 ppm h was proposed for the species to indicate injury onset based on field observations in the Mediterranean Alps [13]. The discrepancy between the calculated CL presented here and previous literature may result from differences in environmental conditions affecting stomatal O₃ uptake between natural forests and the FO₃X experimental facility. Ozone uptake is strongly controlled by stomatal conductance and by environmental drivers such as light, air humidity, temperature, and plant water status [45]. In fact, during summertime, drought often limits stomatal O₃ uptake for trees at the natural forest sites in the Mediterranean Alps (−31% due to soil water deficit, [10]), whereas, at the FO₃X, plants were well-irrigated and any water stress was not expected to reduce g_s for V. lantana and thus enhancing stomatal O₃ uptake. A sensitivity analysis revealed that, if the 30% hypothetical stomatal closure occurred at the FO₃X, the values of POD₁ at the field-observed threshold 12 ppm h AOT40 would be 5.9 to 9.9 mmol m⁻², which agrees with the flux-based CL (7.82 mmol m⁻²) (Table S2). The results indicate that the AOT40-based CL may vary across the target regions due to insufficient consideration of the biological processes underlying O₃ uptake. Given that stomata are the primary interface for O₃ entry into plants, a stomatal flux-based approach should be recommended for a proper setting of CL in forest protection against O₃ pollution.

Interestingly, although a flux-based POD₁ CL for PII was identified as 7.82 mmol m⁻² POD₁, physiological thresholds for a 4% reduction in A_sat and g_s were observed at lower POD₁ values (1.61 mmol m⁻² and 1.22 mmol m⁻², respectively). This suggests that physiological alterations begin before the manifestation of visible injury, supporting the use of combined indicators for a comprehensive assessment. Therefore, a multi-indicator framework combining morphological and physiological indicators is recommended to detect early stress signals and assess cumulative O₃ impact more accurately [46]. This approach improves detection sensitivity, enhances inter- and intra-species comparisons in biomonitoring, and underscores the importance of flux-based metrics in O₃ risk assessment. Low POD values as CLs for physiological parameters were also found in other sensitive species, where the CLs for A_sat were suggested to be 1.9 to 3.5 mmol m⁻² POD₀ for O₃-sensitive deciduous poplar species [47]; therefore, V. lantana can be confirmed as a sensitive species to O₃ [48].

5. Conclusions

This study examined O₃ effects on V. lantana under open-air experimental conditions. Results showed a dose-dependent pattern, with O₃ VFI appearing first in plants exposed to a higher O₃ environment (2.0× treatment). These visual injuries matched field symptoms and were confirmed by colorimetric analysis.

At the same time, physiological responses revealed a more complex pattern. While A_sat and F_v/F_m declined under elevated O₃, g_s and SPAD were less responsive, suggesting that physiological alterations can occur with a different pattern. Notably, thresholds for a 4% reduction in A_sat and g_s were reached at POD₁ levels below those needed to induce VFI, highlighting the importance of combining visual and functional indicators to avoid underestimating damages. The dose–response analysis also showed that the flux-based metric POD₁ better explained variability in physiological parameters than the exposure-based AOT40, supporting the actual consensus that stomatal uptake is a more accurate predictor of plant damage. Thus, we suggested CLs of 1.61 mmol m⁻² and 1.22 mmol m⁻² POD₁ for A_sat and g_s, respectively, and a CL of 7.82 mmol m⁻² for the onset of O₃ VFI.

In conclusion, this study provides the first detailed eco-physiological characterization of the O₃ effect in V. lantana under controlled and realistic exposure conditions. It confirms the species’ moderate sensitivity to O₃ and refines critical thresholds for O₃ risk assessment. V. lantana is confirmed as an promisingbioindicator for integrated O₃ impact monitoring, particularly when physiological and morphological parameters complement visual assessments.