Ozone Response of Leaf Physiological and Stomatal Characteristics in Brassica juncea L. at Supraoptimal Temperatures

: Plants are affected by the features of their surrounding environment, such as climate change and air pollution caused by anthropogenic activities. In particular, agricultural production is highly sensitive to environmental characteristics. Since no environmental factor is independent, the interactive effects of these factors on plants are essential for agricultural production. In this context, the interactive effects of ozone (O 3 ) and supraoptimal temperatures remain unclear. Here, we investigated the physiological and stomatal characteristics of leaf mustard ( Brassica juncea L.) in the presence of charcoal-ﬁltered (target concentration, 10 ppb) and elevated (target concentration, 120 ppb) O 3 concentrations and/or optimal (22/20 ◦ C day/night) and supraoptimal temperatures (27/25 ◦ C). Regarding physiological characteristics, the maximum rate of electron transport and triose phosphate use signiﬁcantly decreased in the presence of elevated O 3 at a supraoptimal temperature (OT conditions) compared with those in the presence of elevated O 3 at an optimal temperature (O conditions). Total chlorophyll content was also signiﬁcantly affected by supraoptimal temperature and elevated O 3 . The chlorophyll a / b ratio signiﬁcantly reduced under OT conditions compared to C condition at 7 days after the beginning of exposure (DAE). Regarding stomatal characteristics, there was no signiﬁcant difference in stomatal pore area between O and OT conditions, but stomatal density under OT conditions was signiﬁcantly increased compared with that under O conditions. At 14 DAE, the levels of superoxide (O 2- ), which is a reactive oxygen species, were signiﬁcantly increased under OT conditions compared with those under O conditions. Furthermore, leaf weight was signiﬁcantly reduced under OT conditions compared with that under O conditions. Collectively, these results indicate that temperature is a key driver of the O 3 response of B. juncea via changes in leaf physiological and stomatal characteristics.


Introduction
Over the next 40 years, the demand for agricultural production is expected to increase by at least 50% as a result of the projected growth of human population [1]. However, agricultural production is highly sensitive to environmental characteristics [2]. According to the latest Intergovernmental Panel on Climate Change (IPCC) Fifth Assessment Report, anthropogenic activities, such as rapid industrial development, have led to substantial climate change due to increased greenhouse gas emission [3]. The global mean temperature is expected to increase by 1.1 • C to 4.8 • C depending on future climate scenarios within this century, which will further worsen global warming and its associated problems related to the ecosystems and crops [3,4]. Climate change is a critical threat to ecosystem health. In particular, global warming may severely damage agricultural crops [4].
To combat global warming, the responses of agricultural crops to increased temperatures in the future must be investigated. Prolonged exposure to elevated temperatures is a critical threat to global crop production [5]. Depending on their developmental stages and specific characteristics, plants respond differently to temperature. However, temperatures exceeding the upper tolerance threshold of plants decrease their net photosynthetic rate and total biomass [6]. Given the importance of temperature in determining net carbon metabolism [7], supraoptimal temperatures can negatively affect plant growth and development. In general, high temperatures increase respiration and photorespiration and possibly deactivate ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) to limit photosynthesis. There are two reasons for the increased photorespiration and suppressed photosynthesis under elevated temperatures. First, high temperature decreases the specificity of RuBisCO for CO 2 relative to that for O 2 , promoting rapid oxygenation. Next, the solubility of O 2 decreases more slowly than that of CO 2 [8]. In addition, the imbalance between photosynthesis and respiration impairs plant growth under elevated temperatures [9].
Furthermore, elevated environmental temperatures can increase stomatal movement, which is linked to primary metabolism [10]. Plant stomatal processes play pivotal roles in carbon cycles [11]. In particular, stomatal conductance is one of the important factors influencing plant net photosynthetic rate and carbon metabolism. However, previous experiments assessing the direct dependence of stomatal conductance on temperature have achieved inconsistent results [12]. In general, stomatal characteristics are strongly affected by elevated temperatures [13]. Recent studies have reported that plants can acclimate to warmer conditions by regulating the interaction between stomatal movement and vapor pressure deficit to allow growth and photosynthesis [14,15]. As a result of these physiological changes, plant growth and development are suppressed at supraoptimal temperatures, which further reduces the total biomass and yield of crops [16].
In addition, tropospheric ozone (O 3 ) concentration is expected to rise along with global warming, since the levels of O 3 precursors, such as NO x , CO, and volatile organic compounds, are predicted to increase in the future [17]. Currently, O 3 is more harmful to agricultural crops than other air pollutants. Specifically, elevated O 3 levels may impede plant physiological processes, including photosynthesis [18]. Plants grown in the presence of elevated O 3 levels show decreased carbon metabolism [19]. In particular, photosynthetic inhibition by O 3 exposure has been attributed to reduced carboxylation efficiency, impaired electron transport, and dysregulated stomatal movement. Consequently, the levels of nonstructural carbohydrates, including sucrose and starch, are reduced [20]. In addition to decreased carbon availability, O 3 -induced stress indirectly affects the carbon balance in plant cells via reactive oxygen species (ROS) generation [21].
The rate of O 3 influx to leaves is controlled by the stomatal aperture. Typically, stomatal closure is recognized as a response to limit O 3 uptake. Acute O 3 exposure can substantially decrease stomatal conductance via ROS accumulation in the guard cells [22].
Several studies using open-top chamber experiments have reported that O 3 decreases stomatal conductance, consequently limiting CO 2 influx to leaves [23,24]. Nonetheless, this mechanism is not supported by the results of other experiments. Some studies have reported that stomata cannot rapidly close as a result of impairment following exposure to high O 3 concentrations [25,26]. Elevated O 3 may delay the stomatal response by delignifying the guard cells and reducing abscisic acid (ABA) sensitivity [26]. The ABA response of stomata is associated with O 3 -induced ethylene emission [27]. Regardless of the mechanisms involved, O 3 is evidently an important determinant of plant physiology.
Individual effects of supraoptimal temperatures and O 3 levels are unlikely to occur in natural environments, because neither factor acts independently on plants [28]. As such, tropospheric O 3 concentration shows a linear relationship with atmospheric temperature [29]. In addition, elevated O 3 is related to increased temperature, which can directly affect the chemical kinetics and mechanisms of O 3 formation [30]. Environmental factors can synergistically affect plant responses, which cannot be predicted based on the results of experiments on individual factors [31]. Although recent studies have proven the individual effects of O 3 or temperature on plants, their interactive effects remain relatively understudied [5,7,19,32]. Furthermore, in addition to greenhouses, agricultural crops are cultivated on open fields, where they are highly prone to exposure to elevated temperatures and O 3 concentrations. Therefore, the interactive effects of these two environmental factors on crops warrant close attention.
To this end, the present study examined the individual and combined effects of supraoptimal temperatures and elevated O 3 concentrations in leaf mustard (Brassica juncea L.), which is widely cultivated in East Asia. Leaf mustard B. juncea is a biennial vegetable crop usually used for edible leaves and seeds to make mustard [33]. The effects of elevated O 3 have been largely researched on Brassica species throughout the world [34][35][36][37]. Singh et al. [37] reported observed decreases in photosynthetic rate and nutrient levels of Brassica campesteris L. var. Kranti under ambient O 3 ranging from 40 to 52 ppb at field condition. Singh et al. [38] also studied the synergistic effects of elevated O 3 and CO 2 on yield and photosynthetic rate of Brassica juncea. There is not sufficient research to assess the effects of O 3 and temperature on the physiological changes of B. juncea. Specifically, we investigated the effects of optimal and supraoptimal temperatures and/or ambient and elevated O 3 concentrations on the physiological and stomatal characteristics of leaf mustard and observed whether supraoptimal temperatures alter the O 3 responses of plants by regulating stomatal movement and carbon metabolism. We hypothesized that (i) the interactive effects of supraoptimal temperature and elevated O 3 concentration on the primary metabolism of leaf mustard are negative and stronger than their individual effects and (ii) supraoptimal temperatures worsen O 3 damage by regulating stomatal movement in B. juncea.

Plant Material and O 3 Fumigation Chamber
Seedlings of leaf mustard (B. juncea), which is widely cultivated in East Asia, were used as the test plant material in this study. Seeds were germinated and cultivated for 2 weeks in a closed-type plant factory (temperature: 20 ± 2 • C; relative humidity: 60 ± 5%; light intensity: 200 ± 20 mol m −2 s −1 ; day length: 16 h) at the University of Seoul, Seoul, Korea (37 • 34 57.5" N, 127 • 03 39.1" E). Thereafter, the seedlings were transplanted into 3 L plastic pots filled with a growing medium containing perlite, vermiculite, and peat moss (Green Partner, Nongwoo Bio, Suwon, Korea). All test seedlings were allowed to acclimatize for a week in the chambers, which were enclosed by glass, under sunlight before starting the treatments.
The present experiment was performed in growth chambers (Growth chamber, Koito Industries, Yokohama, Japan) equipped with an O 3 generator (ON-1-2, Nippon Ozone Co., Tokyo, Japan). The fumigation system has been described elsewhere [39]. For each test, 15 plants with similar growth conditions were placed in a control chamber with a charcoal filter, and an additional 15 plants were placed in a treatment chamber with an O 3 fumigator. All plants were irrigated well and randomly placed everyday throughout the experiment. Leaves were sampled twice at 7 and 14 days after the beginning of exposure (DAE) between 09:00 and 12:00 h. The experiment lasted from March to April 2018. Fully expanded leaves were used to analyze the O 3 response of physiological and stomatal characteristics. Samples were stored at − 80 • C until analysis.
The treatment conditions in the growth chamber are summarized in Table 1. The elevated O 3 concentration used was based on the hourly average maximum O 3 concentration from March to July 2016 measured in South Korea [40]. The optimal temperature was 22 • C, since Brassica spp. grow the best within the temperature range of 22-25 • C according to previous reports [41][42][43]. The supraoptimal temperature was 5 • C above the optimal temperature range for leaf mustard considering one of the moderate scenarios for 2100 projected previously [3]. The test plants were exposed to O 3 for 8 h daily from 09.30 to 17.30 h while controlling the temperature and relative humidity for 24 h (Figure 1). Values are presented as mean ± SD (n = 5).

Physiological Characteristics
Photosynthetic rate curves were plotted against intercellular CO 2 concentration (A/C i ) using a portable photosynthesis measurement system (Li-6400 XT, LI-COR Inc., Lincoln, NE, USA) with an LED source (6400-02B, LI-COR Inc., Lincoln, NE, USA). Measurements were performed on the second to fourth fully expanded leaves in each treatment group, with CO 2 concentration settings of 50, 100, 150, 200, 400, 600, 800, 1000, and 1200 µmol mol −1 , tested under photosynthetically active radiation of 1000 µmol m −2 s −1 , block temperature of 20 • C, and relative humidity of 50-60%. The maximum reaction velocity of RuBisCO for carboxylation (V cmax ), maximum rate of electron transport (J max ), triose phosphate use (TPU), daytime respiration (R d ), and mesophyll conductance (g m ) were calculated based on the response curves using the A/C i curve fitting utility program described previously [44].
Chlorophyll a (Chl a) fluorescence was determined for the second to fourth fully developed leaves using Pocket PEA software (PEA plus V1.10, Hansatech Instrument Ltd., Norfolk, UK) between 10.00 and 12.00 h. Before measurement, leaves were adapted to darkness using leaf clips for 30 min. A saturating pulse at an intensity of 3500 µmol m −2 s −1 (peak wavelength, 627 nm) was applied to the upper surface of the test leaves to measure the minimum fluorescence (F o ), maximum fluorescence (F m ), and OJIP transients. The maximum photochemical efficiency of photosystem II (PS II) was determined as the ratio of variable fluorescence (F v The measured values of OJIP transients indicate F o intensity at 50 µs when all PS II reaction centers (RCs) are open (O-step), fluorescence intensity at 2 ms (J-step), fluorescence intensity at 30 ms (I-step), and F m intensity when all PS II RCs are closed (P-step). The JIP parameters were calculated to identify the extent of damage to the electron acceptor sites of PS II by O 3 according to the JIP test equations [45,46]. The value of each JIP parameter is listed in Table S1.

Chlorophyll Content
Chl a, chlorophyll b (Chl b), and total chlorophyll (Chl a + Chl b) content was estimated as previously described [47]. Fresh leaves (0.1 g) were extracted in 10 mL of 80% (v/v) acetone for 14 days at 4 • C. The chlorophyll content was quantified using a microplate reader (Epoch Microplate Spectrophotometer, Synergy, BioTek, Winooski, VT, USA) at an absorbance (A) of 663, 645, and 470 nm. Chlorophyll content was determined using the following formulas: In practice, the phytol chain of chlorophyll molecules can be easily cleaved with the addition of 80% acetone [48]. We did not consider the phytol chain cleavage, because phytol has the same absorption and light spectra as chlorophyll and thus does not affect the values obtained using this method [49].

Stomatal Characteristics
For the measurement of stomatal characteristics, leaf samples were harvested at 14 DAE between 09:00 and 12:00 h and freeze-dried using a lyophilizer (FD 8508, ilShinbiobase CO. Ltd., Dongducheon, South Korea). Stomatal density per leaf and stomatal size were evaluated via field emission scanning electron microscopy (FESEM; SU-70, Hitachi, Tokyo, Japan). Stomatal density was determined based on the number of stomata obtained by FESEM.

Hydrogen Peroxide (H 2 O 2 ) and Superoxide (O 2 − ) Accumulation
H 2 O 2 and O 2 accumulation in sampled leaves was measured as described previously [50], with slight modification. To detect H 2 O 2 , leaf discs were cut with a cork borer (diameter, 2 cm), vacuum-infiltrated in 1 mg ml −1 of 3,3-diaminobenzidine (DAB) in 0.2 M HCl (pH 3.8), and incubated at 25 • C for 4 h in the dark. To detect O 2 -, the leaf discs were cut (diameter, 2 cm), vacuum-infiltrated in 50 mM potassium phosphate buffer (pH 7.8) containing 0.1% (w/v) nitroblue tetrazolium (NBT), and incubated at 25 • C for 20 min in the dark. For both measurements, leaf discs were immersed in 96% (v/v) ethanol for 20 min at 70 • C to remove chlorophyll. After cooling, the leaf discs were stored in 70% (v/v) glycerol. The leaf discs were photographed and observed under a stereomicroscope (Leica M275, Leica Microsystems, Mannheim, Germany). The stained areas were calculated as the proportion of pixels in the stained area to the total pixels using Adobe Photoshop CS6 (Adobe Inc., Mountain View, CA, USA).

Growth Characteristics
All plants in each treatment chamber were collected at 14 DAE. Five plants per treatment were randomly selected to determine leaf fresh and dry weight, leaf number, and leaf area. Leaf area was measured on three fully expanded leaves of five plants using winFolia (Regent Instruments Inc., Sainte-Foy, QC, Canada). Leaf dry weight was obtained by drying the samples for 48 h at 60 • C in an oven (HK-300DO, HUKO FS, Seoul, Korea).

Statistical Analysis
Effects of O 3 concentration, temperature, sampling date, and their interactions on the physiological and stomatal characteristics of B. juncea were analyzed using two-way or three-way analysis of variance (ANOVA). Tukey's honestly significant difference test (p ≤ 0.05) was used to compare the differences among the parameters tested. Significance of differences in values at 7 and 14 DAE among various treatments was tested using independent t-test. All analyses were performed using SPSS Statistics 25 (SPSS Inc., Chicago, IL, USA). Values in figures and tables are presented as mean ± SD.

A/C i Curve Response
At 7 DAE, V cmax was significantly decreased under ambient O 3 + supraoptimal temperature (T) (by 19.3%) and elevated O 3 + supraoptimal temperature (OT) (by 36.2%) conditions compared with that under ambient O 3 + optimal temperature (C) conditions. At 14 DAE, V cmax under O and OT conditions was significantly reduced by respectively 44.6% and 54.3% compared with that under C conditions. Therefore, supraoptimal temperatures did not significantly affect the V cmax of B. juncea at 14 DAE ( Figure 2). Three-way ANOVA showed that V cmax was significantly affected by all individual factors and their interactions except for the temperature × O 3 × sampling date interaction (Table S2).
At 7 and 14 DAE, J max under OT conditions was significantly decreased by respectively 34.9% and 67.9% compared with that under C conditions. Furthermore, J max under O conditions was significantly reduced by 55.05% compared with that under C conditions ( Figure 2). Three-way ANOVA showed that two individual factors, namely elevated O 3 and sampling date, significantly affected J max . Moreover, the interactions among factors also significantly affected J max , except the temperature × O 3 × sampling date interaction (Table S2).
At 7 DAE, there were no significant differences in TPU under O, OT, and T conditions compared with that under C conditions; however, at 14 DAE, TPU under O (by 31.6%) and OT (by 43.4%) conditions was significantly reduced compared with that under C conditions ( Figure 2). Three-way ANOVA showed that TPU was significantly affected by two individual factors, namely elevated O 3 and sampling date, as well as by interactions among all factors, except the temperature × O 3 × sampling date interaction (Table S1).
At 7 DAE, R d was increased under OT conditions compared with that under C and O conditions (by 56.3% and 53.3%, respectively). At 14 DAE, R d under T conditions was significantly increased (by 44.8%) compared with that under C conditions ( Figure 3). Three-way ANOVA showed that elevated temperature strongly affected R d (Table S2).  Moreover, g m was significantly reduced under OT conditions compared with that under C conditions. Similarly, g m under OT conditions was significantly reduced by 47.4% compared with that under T conditions (Figure 3). Three-way ANOVA indicated that g m was significantly affected by the sampling date alone (Table S2).

Chl Content
At 14 DAE, total Chl content was significantly decreased under O (by 23.2%) and OT (by 55.3%) conditions compared with that under C conditions. However, at 7 DAE, total Chl content was significantly reduced under OT (by 36.2%) conditions alone (Figure 4). Total Chl content was affected by two individual factors, namely supraoptimal temperature and O 3 , whereas sampling date and interactions among all factors did not significantly affect total Chl content, except the O 3 × sampling date interaction (Table S2).
Chl a/b ratio was significantly reduced under OT conditions compared with that under C conditions at both 7 (by 25.0%) and 14 (by 25.7%) DAE (Figure 4). Three-way ANOVA showed no significant effect of any factor or interaction on this ratio except for the temperature × sampling date interaction (Table S2).

Chl a Fluorescence
In the OJIP transient curves of B. juncea, each step showed a different response to various treatments. Overall, the expected degradation of the photosynthetic apparatus in plants grown under O and OT conditions was observed in the OJIP transient curves. Fluorescence at the J-step was significantly reduced under OT conditions compared with that under C conditions at 7 and 14 DAE (by 12% and 23.7%, respectively). Fluorescence at the I-step was also significantly decreased under OT conditions compared with that under C conditions at 14 DAE (by 28.8%). Moreover, relative fluorescence at the P-step was decreased under O and OT conditions (by 25% and 33%, respectively) compared with that under C conditions at 14 DAE ( Figure 5B). The values of JIP parameters estimated based on the OJIP transient curves of Chl a fluorescence were significantly different between the test and controls plants, and these values were used to construct the spider plots shown in Figure 6. There were no significant differences among treatments in terms    Table S1

Stomatal Characteristics
The stomatal appearance differed among plants exposed to supraoptimal temperature, elevated O 3 concentration, or a combination of both ( Figure 7A). Stomatal density on the abaxial leaf surface in B. juncea was decreased (by 45%) under O conditions compared with that under C conditions. However, there were no significant differences in stomatal density under T, OT, and C conditions ( Figure 7B). The stomatal pore area was slightly increased by supraoptimal temperatures, albeit not significantly. The stomatal pore area was significantly decreased under O (by 57.8%) and OT (53.9%) conditions compared with that under C conditions ( Figure 7B). Two-way ANOVA showed that individual factors and their interactions significantly affected stomatal density, whereas only ambient and elevated O 3 concentration significantly affected stomatal pore area (Table S2).

H 2 O 2 and O 2
− Accumulation DAB and NBT staining showed ROS accumulation in B. juncea at 14 DAE ( Figure 8A). The DAB-stained area, representing H 2 O 2 accumulation, was significantly increased under O and OT conditions compared with that under C conditions. However, there were no significant differences between C and T conditions at 14 DAE ( Figure 8B). In addition, there were no significant differences between O and OT conditions.  (Table S2).

Plant Growth Characteristics
Leaf fresh weight was significantly decreased under T, O, and OT (by 26.5%, 57.3%, and 93.4%, respectively) conditions compared with that under C conditions. Leaf fresh weight under OT conditions was significantly decreased compared with that under O conditions. Conversely, leaf dry weight was not significantly different between OT and O conditions (Figure 9). Leaf dry weight was significantly decreased under T, O, and OT (by 34.7%, 48.3%, and 55.8%, respectively) conditions compared with that under C conditions. Leaf number and area were significantly decreased under O and OT conditions compared with those under C conditions (Figure 9). However, there were no significant differences between O and OT conditions. Two-way ANOVA showed that leaf fresh and dry weight was significantly affected by all individual factors and their interactions, whereas leaf number and area were not affected by any factor or interaction (Table S2).

Discussion
Given the potential effects of supraoptimal temperature and elevated O 3 on plants, the present study explored the impact of these environmental factors on the physiological and stomatal characteristics of B. juncea. We demonstrated that the interactions of supraoptimal temperature and elevated O 3 negatively affected leaf mustard rather than the individual factors.
Regarding physiological characteristics, B. juncea leaves showed lower photosynthetic capacity under OT conditions than under other conditions, as indicated by V cmax , J max , and TPU. Measurements of V cmax and J max , which are indicators of biochemical capacity, are typically based on intercellular CO 2 . V cmax decreased under O and OT conditions compared with that under C conditions at both 7 and 14 DAE (Figure 2). This result is consistent with previous reports on the effects of O 3 on plant physiological characteristics [51,52]. Previous research found a significant reduction in net photosynthesis (P n ) of B. juncea under O and OT conditions compared to C at 7 and 14 DAE. Furthermore, there was also significant difference in P n between O and OT conditions at 14 DAE [39]. V cmax indicates the intrinsic photosynthetic capacity as well as RuBisCo activity and kinetics, and J max indicates the rate of RuBP regeneration via electron transport [44,53]. Biochemical limitations related to RuBisCo are associated with reduced CO 2 assimilation in the presence of elevated O 3 [54,55]. In general, O 3 reduces the RuBisCo protein content of plants by producing ethylene [56]. O 3 stress impairs the photosystems (PSs), thereby generating the energy required to produce NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate) [57]. Photosynthetic capacity is linked to leaf nitrogen (N) content since the majority of leaf N comes from thylakoid proteins and stromal enzymes [58]. O 3 can result in lower N content per leaf area, higher investment of N in cell walls, and a reduction of leaf N allocation to photosynthetic processes [59]. However, in the present study, there were no significant differences in V cmax between C and T conditions at 14 DAE (Figure 2). This result suggests that supraoptimal temperature (5 • C above the optimal) did not affect V cmax as much as elevated O 3 did. The same tendency was observed for J max and TPU. Similarly, Thwe et al. [57] have reported a strong correlation between V cmax and J max in tomato under O 3 stress. However, some previous studies have reported that a minor limitation to RuBisCo activation is closely related to moderately high temperatures [60,61]. Indeed, in the present study, J max and TPU were not significantly affected by supraoptimal temperatures alone (Table S2). Increased J max and TPU were observed under T conditions compared with those under C conditions ( Figure 2). By shifting the operating range beyond the optimal temperature, short-term supraoptimal temperature of about 5 • C may decrease P n [62]. In previous study, P n of B. juncea under elevated temperature was significantly decreased compare to that in optimum temperature at 7 DAE [39]. This form of acclimation, which results in a general up-regulation of leaf N and photosynthetic proteins, is based on the availability of N resources for higher investment in the photosynthetic apparatus [62]. Under sustained high temperatures, most plants can acclimate their photosynthetic traits. Through thermal acclimation, metabolic activities are modified to compensate for temperature changes, resulting in metabolic homeostasis [63]. Three-way ANOVA on the response parameters of A/Ci curves indicated that R d was significantly affected by supraoptimal temperature. The rates of enzymatic processes involved in R d are enhanced by high temperatures [64]. An increase in R d along with a decrease in net photosynthesis reduces carbohydrate availability to plants [65]. In this study, there were no significant differences in these parameters in the presence of elevated O 3 . Contrary to our results, Calatayud et al. [51] reported increased R d in a Mediterranean endemic plant exposed to an O 3 concentration of 70 ppb. In this study, significant reductions in g m were observed under O conditions at 14 DAE and under OT conditions at both 7 and 14 DAE compared with the values under C conditions ( Figure 3). Several studies on soybean, birch, and poplar have reported negative effects of elevated O 3 on g m [66][67][68]. As such, g m is affected by O 3 conductance through cell wall, intercellular air spaces, and intracellular fluid [69]. Elevated O 3 likely reduces g m via mechanisms involving the altered levels, shape, and position of the chloroplasts and thickening of the cell wall [70]. g m is closely correlated with stomatal conductance (g s ), since CO 2 and H 2 O are likely to share a common pathway in leaves [71]. In a previous study, a result was that a rapid transient decrease in g s can occur when O 3 level was elevated as with g m . The g s of B. juncea under OT condition was found to be significantly lower than that of plants under T condition [39].
Reduced total Chl content is considered an indicator of O 3 -induced biochemical damage [72]. In this study, total Chl content was decreased under O and OT conditions compared with that under C conditions at 14 DAE. Moreover, total Chl content under OT conditions was lower than that under T and O conditions at 14 DAE (Figure 4). A decrease in the total Chl content of leaves following O 3 exposure has been reported previously [73,74]. O 3 indirectly affects plant Chl content by accelerating leaf senescence [75]. A decrease in total Chl content generally reduces CO 2 assimilation [76], and there is a strong correlation between total Chl content and photosynthetic rate [77].
A significant decrease in the Chl a/b ratio was observed under OT conditions compared with that under C conditions at 7 and 14 DAE (Figure 4). These results regarding the relative O 3 sensitivities of Chl a and b are overall contrary to previous reports [78]. There was a positive correlation between the ratio of Chl a to b and the ratio of PS II to light-harvesting chlorophyll a/b protein complex-II (LHCII) level. LHCII is the major light-harvesting complex in plants that exclusively contains Chl b and, accordingly, has a low Chl a/b ratio [79]. Czuba and Ormrod [80] reported that the Chl b content of Lepidium sativum L. was decreased to a greater extent than its Chl a content by O 3 . However, greater reductions in Chl a content than in Chl b content by O 3 have also been reported in other plants [81].
In this study, OJIP transient curves were plotted and compared as indicators of photoinhibition in leaves under all treatments. Photoinhibition decreases the photosynthetic activity by reducing light-induced carbon assimilation [82]. Overall, the slope of OJIP curve significantly decreased from the J-to P-step under OT conditions at 7 and 14 DAE, whereas this decrease was only observed from the I-to P-step at 14 DAE ( Figure 5A). These results indicate that the function of PS II RCs was more severely inhibited under OT conditions than under O conditions. Marzuoli et al. [83] reported that elevated O 3 levels significantly decreased PS II photochemical efficiency and electron flow in lettuce. O 3 induced ROS damage PS II RCs [84]. According to the OJIP transient curves in this study, elevated temperature did not negatively affect PS II RCs. However, ϕP O was significantly decreased under OT conditions at both 7 and 14 DAE ( Figure 6). This result is consistent with previous reports on the effects of elevated O 3 [57,85]. The observed ϕP O reduction may be a result of enhanced non-photochemical processes of PS II light-harvesting antennae, which are related to the suppression of photochemical quenching and photodamage of PS II RCs [86]. In addition, ϕD O was increased under O and OT conditions at 7 and 14 DAE ( Figure 6). The observed increase in ϕD O (ϕD O = F o /F m ) was reflected in the increased value of F o , which was perhaps due to the inactivation of some PS II RCs [87].
Stomata play a crucial role in determining the O 3 influx to leaves, because the majority of the O 3 enters the leaf via stomatal pores [88]. Since O 3 enters leaves of the plant through stomata, O 3 influx are highly dependent on stomatal density and conductance [89]. At 7 DAE, O 3 influx of B. juncea under OT condition was significantly higher than plants grown under O condition, as g s significantly increased under OT compared to those in O [39]. Elevated O 3 -induced oxidative stress also results in ultrastructural changes to mesophyll cells and their cell walls in plants, limiting O 3 entering into the leaves [90]. In a previous study, 100 ppb of O 3 led to the collapse of some epidermal cells adjacent to the stomata, and 150 ppb of O 3 led to the complete collapse of epidermal cells and loss of leaf structural integrity [91]. Consistent with previous reports, we observed a collapse of stomatal shape under O conditions ( Figure 7A). Moreover, the stomatal density in leaf mustard under O conditions was significantly lower than that under other conditions at 14 DAE ( Figure 7B). Neufeld et al. [92] reported that 100 ppb of O 3 tended to reduce stomatal density, suggesting structural changes in plants. Reductions in stomatal width and pore area in the presence of elevated O 3 have been confirmed in previous studies [93,94]. The stomatal density of B. juncea was affected by supraoptimal temperatures, elevated O 3 , and their interaction, while its stomatal pore area was only affected by elevated O 3 (Table S2). Some previous studies have reported increased stomatal densities at supraoptimal temperatures [95,96]. Some plant species can develop leaves with different stomatal density in supraoptimal temperature, which can affect g s [97]. g s of B. jnucea under T and OT condition was significantly higher that in the C and O condition at 7 DAE, respectively. However, no significant difference in g s between C and T condition was observed at 14 Figure S1). The first O 3 injury symptoms on the surface of leaves under OT conditions at 3 DAE and those under O at 5 DAE were reported [39]. A major effect of O 3 on plants is ROS generation. ROS production in plant cells is closely related to the O 3 flux and oxidative stress tolerance of plants [98,99]. ROS production is a critically harmful process and is also a key component of the abiotic stress responsive signaling networks of plants [100]. In this study, DAB  produces a strong effect on carbon assimilation, and it is a vital precursor of other ROS as an unstable radical with a high redox potential [101]. H 2 O 2 can be produced from O 2 via Mn-SOD [102].
In this study, we focused on the leaf growth response of B. juncea, because it is one of the most widely consumed leafy vegetables in East Asia. A significant decrease in leaf fresh and dry weight under O and OT conditions was observed (Figure 9). Many studies have indicated that prolonged O 3 exposure is typically detrimental to leaf growth and development [103], which is possibly due to O 3 -induced damage to mesophyll cells upon uptake into the plants [104]. Li et al. [19] reported that the growth of eggplant (Solanum melongena L.) was significantly reduced in the presence of elevated O 3 . Cell death as well as suppression of photosynthetic and stomatal activity in leaves can decrease the biomass and yield of leafy vegetables [105]. Additionally, leaf area was significantly reduced under O and OT conditions ( Figure 9). The suppression of leaf primary metabolism leads to a reduction in leaf area [18].

Conclusions
By studying the responses of leaf physiological and stomatal characteristics, we demonstrated that elevated O 3 caused more severe damage in leaf mustard (B. juncea) at supraoptimal temperatures than at optimal temperatures. Stomatal density, which is related to O 3 influx to leaves, was higher in the presence of elevated O 3 at supraoptimal temperatures than that at optimal temperature, indicating detrimental effects of O 3 on the physiological, biochemical, and growth characteristics of B. juncea at 14 DAE. In particular, the interactive effects of O 3 stress and supraoptimal temperature decreased photosynthetic parameters, including J max and TPU. Furthermore, Chl a fluorescence at the J-step of the OJIP transient curves was decreased in the presence of elevated O 3 at supraoptimal temperatures. Regarding biochemical characteristics, NBT-stained spots indicating O 2 accumulation in plant cells were abundant under OT conditions. As a consequence of physiological and biochemical responses, elevated O 3 at supraoptimal temperatures significantly reduced leaf fresh and dry weight. Collectively, these findings indicate the interactions of supraoptimal temperature and elevated O 3 worsen the damage to B. juncea rather than the individual factors.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/land10040357/s1, Table S1: Formulas and descriptions of OJIP parameters based on data obtained from OJIP transient curves. Table S2: Results of analyses of variance of the main effects of temperature, O 3 , and sampling date and their interactions on A/C i curve response, chlorophyll content, chlorophyll a fluorescence, stomatal density, stomatal pore area, hydrogen peroxide and superoxide radical accumulation, and leaf growth parameters. Table S3

Conflicts of Interest:
The authors declare no conflict of interest.