*Article*

#### **Light Energy Partitioning under Various Environmental Stresses Combined with Elevated CO 2 in Three Deciduous Broadleaf Tree Species in Japan**

**Mitsutoshi Kitao 1,\*, Hiroyuki Tobita 2, Satoshi Kitaoka 2, Hisanori Harayama 1 , Kenichi Yazaki 2, Masabumi Komatsu 2, Evgenios Agathokleous 3 and Takayoshi Koike 4**


Received: 28 March 2019; Accepted: 30 May 2019; Published: 3 June 2019

**Abstract:** Understanding plant response to excessive light energy not consumed by photosynthesis under various environmental stresses, would be important for maintaining biosphere sustainability. Based on previous studies regarding nitrogen (N) limitation, drought in Japanese white birch (*Betula platyphylla* var. *japonica*), and elevated O3 in Japanese oak (*Quercus mongolica* var. *crispula*) and Konara oak (*Q. serrata*) under future-coming elevated CO2 concentrations, we newly analyze the fate of absorbed light energy by a leaf, partitioning into photochemical processes, including photosynthesis, photorespiration and regulated and non-regulated, non-photochemical quenchings. No significant increases in the rate of non-regulated non-photochemical quenching (JNO) were observed in plants grown under N limitation, drought and elevated O3 in ambient or elevated CO2. This suggests that the risk of photodamage caused by excessive light energy was not increased by environmental stresses reducing photosynthesis, irrespective of CO2 concentrations. The rate of regulated non-photochemical quenching (JNPQ), which contributes to regulating photoprotective thermal dissipation, could well compensate decreases in the photosynthetic electron transport rate through photosystem II (JPSII) under various environmental stresses, since JNPQ+JPSII was constant across the treatment combinations. It is noteworthy that even decreases in JNO were observed under N limitation and elevated O3, irrespective of CO2 conditions, which may denote a preconditioning-mode *adaptive response* for protection against further stress. Such an *adaptive response* may not fully compensate for the negative e ffects of lethal stress, but may be critical for coping with non-lethal stress and regulating homeostasis. Regarding the three deciduous broadleaf tree species, elevated CO2 appears not to influence the plant responses to environmental stresses from the viewpoint of susceptibility to photodamage.

**Keywords:** chlorophyll fluorescence; drought; elevated O3; N limitation; non-photochemical quenching; photodamage

#### **1. Introduction**

Although light is essential for plant growth, plants can su ffer from excessive light, especially when combined with other environmental stresses. Light energy absorbed by a leaf is mainly consumed

*Climate* **2019**, *7*, 79

by photochemical processes such as electron flow to photosynthesis, photorespiration and alternative pathways [1].

Conversely, absorbed light energy is also dissipated by non-photochemical processes divided into two parts: Constitutive, non-regulatory, non-photochemical quenching, and regulatory light-induced, non-photochemical quenching [2–5]. When photosynthetic electron transport is suppressed under environmental stresses, an increase in the fraction of non-regulatory, non-photochemical quenching suggests that plants cannot fully dissipate excess energy through a regulated process [5–9]. Non-regulated, non-photochemical quenching consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of 1O2 via the triplet state of chlorophyll ( 3chl\*) [10–13]. 1O2 can lead to PSII photodamage directly, or via inhibiting PSII repair processes [14–16]. Non-regulated, non-photochemical quenching can be a measure of oxidative stress, as the level of lipid peroxidation indicated by malondialdehyde (MDA) accumulation was closely correlated with the quantum yield of non-regulated, non-photochemical quenching (Y(NO)) in *Arabidopsis thaliana* under a water deficit imposed by withholding the water supply [17].

Plants can acclimate to various environmental conditions by adjusting their leaf physiological characteristics to prevent photodamage [18,19]. For example, within a canopy, sun leaves grown under higher irradiance have a higher photosynthetic capacity with higher area-based leaf nitrogen (Narea) than shade leaves [20], a mechanism contributing to maximize photosynthetic carbon gain at the whole plant level by utilizing limited nitrogen optimally [21]. The net photosynthetic rate is known to be proportional to Narea, since an increase in Narea suggests an increase in Rubisco, a major photosynthetic enzyme [22]. As Rubisco is a key enzyme catalyzing both photosynthesis and photorespiration, electron flow through PSII consumed by the processes also increases with increasing Narea [23]. As energy dissipation through photosynthetic electron transport is closely related to Narea [23], such Narea-related photosynthetic acclimation along the light gradient within the canopy, can also contribute to suppress the risk of photodamage in response to the maximum irradiance during sunflecks, in combination with xanthophyll-related photoprotective energy dissipation [24,25].

Environmental stresses such as N limitation, drought and elevated O3, causing a reduction in photosynthesis, would increase excessive light energy via a reduction in photosynthetic electron consumption, since photosynthetic carbon assimilation needs NADPH, generated via electron transport [26]. In the coming future, environmental stresses such as nitrogen limitation, as a relative constraint on plant-growth enhancement under elevated CO2 [27], drought [28], and high O3 exposure [29–31], are predicted to occur more frequently under global warming and elevated CO2 concentrations.

We hypothesized that, even under various environmental stresses such as N limitation, drought and elevated O3 under CO2 enrichment in the coming future, the non-regulated, non-photochemical quenching should be kept under a certain level, to prevent photosynthetic apparatus from oxidative damage [14–16]. This is achieved by a functional coordination of energy dissipation primarily through N-required electron transport [23], and complementarily through xanthophyll-related thermal energy dissipation, which does not require N investment [8].

To test the hypothesis, we newly analyzed data from previously published works from our research group using three deciduous broadleaf tree species [32–34], where the response of plants to di fferent environmental factors was assessed by chlorophyll fluorescence, so as to assess the fate of absorbed light energy consumed by photochemical processes, and dissipated through constitutively non-regulatory and regulatory light-induced, non-photochemical quenching. We also assessed the light energy not absorbed by a leaf, involved in a bulk loss in chlorophyll pigments, which also has a protective role against photodamage via a reduction in absorbed light energy.

#### **2. Materials and Methods**

This study is in part a collective re-analysis of previously published data [32–34]. Regarding "N limitation under elevated CO2" and "drought under elevated CO2", regulated

and non-regulated non-photochemical quenchings were newly calculated based on data from the previous studies [32,33]. Conversely, regarding "elevated O3 under elevated CO2", all data except for An were not published previously (cf. [34]).

#### *2.1. N Limitation under Elevated CO2*

Data of Japanese white birch (*Betula platyphylla* var. *japonica*) seedlings grown under limited N and elevated CO2 were obtained from the study by Kitao et al. [32]. Experiments of N limitation under elevated CO2 were conducted using a natural daylight phytotron (26/16 ◦C, day/night; ca. 90% of full sunlight) in Hokkaido Research Center, Forestry and Forest Products Research Institute (FFPRI) in Sapporo, Japan (43◦N, 141◦E; 180 m above sea level). Details are described in Kitao et al. [32]. One-year-old seedlings of Japanese white birch (*Betula platyphylla* var. *japonica*), a pioneer tree species, 15 to 20 cm in height, were transplanted in free-draining plastic pots filled with clay loam soil mixed with Kanuma pumice soil (1:1 in volume). Pots were placed on trays to prevent nutrient drainage. Each of two CO2 treatments: 360 μmol mol−<sup>1</sup> (ambient CO2 treatment, A-CO2); and 720 μmol mol−<sup>1</sup> (elevated CO2 treatment, E-CO2) were replicated in two chambers. Two nitrogen levels were applied: 700 mg per plant (adequate nitrogen, +N), or 100 mg per plant (limited nitrogen, -N). The former treatment was conducted as 100 mg N pot−<sup>1</sup> week−<sup>1</sup> for 7 weeks during CO2 treatment, whereas the latter one was conducted as 100 mg N pot−<sup>1</sup> only once at the onset of CO2 treatment. We supplied relatively high N for +N treatment to provide adequate N to plants, so as to reach their normal state relative to nursery-grown seedlings. Area-based leaf N (Narea) in the seedlings grown in +N treatment was comparable to those grown in the nursery of FFPRI (data not shown). Conversely, we supplied substantially low N for -N treatment, expecting photosynthetic down-regulation under N limitation [35].

#### *2.2. Drought under Elevated CO2*

Data of Japanese white birch seedlings grown under limited water supply and elevated CO2 were obtained from the study by Kitao et al. [33]. Experiments of drought under elevated CO2 were also conducted for 1-year-old seedlings of Japanese white birch in the phytotron in Hokkaido Research Center, FFPRI, as described above. Details are described in Kitao et al. [33]. Each of the two CO2 treatments i.e., 360 (ambient CO2 treatment: A-CO2) and 720 μmol mol-<sup>1</sup> (elevated CO2 treatment: E-CO2) was replicated in three chambers. Six randomly selected seedlings in each chamber were supplied daily with 100 mL of water or nutrient solution (once per week) (adequate water supply), while the other six seedlings (totally 12 seedlings) received only 100 mL of nutrient solution once weekly (drought). Each plant received a total of 100 mg N during the experiment, which corresponded to limited N treatment, as described above. The lowest predawn leaf water potential (i.e., measured just prior to the scheduled watering), which was in equilibrium with the soil water potential, was A-CO2 + adequate water supply: −0.13, A-CO2 + drought: −0.52, E-CO2 + adequate water supply: −0.12 and E-CO2 + drought: −0.39 MPa [33]. The values of water potential in the drought treatment were moderate, since no wilting in the seedlings was observed. Leaves flushed and developed during the drought treatment were used for the measurements.

#### *2.3. Elevated O3 under Elevated CO2*

Data of Japanese oak (*Quercus mongolica* Fisch. ex Ledeb. var. *crispula* (Blume) H. Ohashi) and Konara oak (*Q. serrata* Murray) seedlings grown under elevated O3 and CO2 were obtained from the study by Kitao et al. [34]. Experiments of elevated O3 under elevated CO2 were conducted in a free-air concentration-enrichment (FACE) exposure system, consisting of 12 plots (3 replicates per treatment), located at the nursery of FFPRI in Tsukuba, Japan (36◦00'N, 140◦08'E, 20 m a.s.l.).

Details are described in Kitao et al. [34]. One-year-old seedlings of Japanese oak and Konara oak, gap-dependent mid-successional tree species, approximately 5 cm in height under dormancy, were transplanted directly to the ground in the plots. The treatments were as follows: Control (unchanged ambient air), elevated CO2 (Target set, 550 μmol mol−1), elevated O3 (Target set, twice-ambient), and elevated CO2 + O3 (550 μmol mol−<sup>1</sup> CO2 and twice-ambient O3). Plants were grown under the treatments for two growing seasons. Measurements of gas exchange and chlorophyll fluorescence were conducted in the second growing season.

#### *2.4. Measurements of Gas Exchange and Chlorophyll Fluorescence*

Measurements of gas exchange and chlorophyll fluorescence were conducted with a portable photosynthesis measuring system (Li-6400, Li-Cor, Lincoln, NE, USA), combined with a portable fluorometer (PAM-2000, Walz, E ffeltrich, Germany) for plants grown under "N limitation with CO2 enrichment", or a leaf chamber fluorometer (Li-6400-40, Li-Cor) for plants grown under "drought, and elevated O3 under elevated CO2". Details are described in Kitao et al. [32–34]. The net photosynthetic rate (An), quantum yield of PSII electron transport (Y(II)), quantum yield of non-regulate, non-photochemical quenching in PSII (Y(NO)), and finally the quantum yield of regulated, non-photochemical quenching in PSII (Y(NPQ)) [2–5] were measured at a photosynthetic steady state under saturating light intensities provided by a red/blue LED array (Li-6400-40, Li-Cor), with blue light comprising 10% of the total PPFD. We measured Y(NO) and Y(NPQ), based on the simple approach: Y(NO) = F/Fm, and Y(NPQ) = F/Fm' − F/Fm, where F, F m and F m' is the relative fluorescence yield at steady state illumination, the relative maximum fluorescence yield in dark-adapted conditions, or that during illumination, respectively [4,5]. Regarding the data sets of drought and elevated O3 under elevated CO2, we measured F, F m' and Fo' (the minimum fluorescence yield during illumination) during the gas exchange measurements, but did not measure F m. We measured Fv/Fm on the following day, after an overnight dark-adaptation in the same leaves for the gas exchange, and chlorophyll fluorescence measurements with the photosynthesis system (Li-6400, Li-Cor) for drought-treated plants [33], and with a portable fluorometer (Mini-PAM, Walz) for O3-treated plants [34]. Since Fo' is estimated as Fo' = Fo/(Fv/Fm + Fo/Fm') [36], F m can be estimated as F m = (F m' × Fo' × Fv/Fm)/((F m' − Fo') × (1 − Fv/Fm)). This would be a practical approach to determine F m for many samples in the field after the fluorescence measurements during daytime. Leaf absorptance (ABS) was calculated from a calibration curve between SPAD readings (measured with a SPAD chlorophyll meter, SPAD 502, Minolta, Osaka, Japan) and leaf absorptance [32–34]. Based on the chlorophyll fluorescence parameters, the electron transport rate (JPSII) was calculated as JPSII = Y(II) × ABS × light intensity × 0.5 [6]. Analogous to JPSII, the rate of regulatory thermal dissipation (JNPQ) and the rate of non-regulatory energy dissipation via heat or fluorescence (JNO) were estimated from Y(NPQ) × ABS × light intensity × 0.5 and Y(NO) × ABS × light intensity × 0.5, respectively [4]. Light energy not absorbed by chlorophyll in a leaf (JChl) was estimated as JChl = (1 − ABS) × light intensity × 0.5.

#### *2.5. Leaf N Content*

Regarding '*elevated O3 under elevated CO2'*, the leaves were sampled after the measurements and used for a determination of Narea by the combustion method, using an analysis system composed of an N/C determination unit (SUMIGRAPH, NC 800, Sumika Chem. Anal. Service, Osaka, Japan), a gas chromatograph (GC 8A, Shimadzu, Kyoto, Japan), and a data processor (Chromatopac, C R6A, Shimadzu).

#### *2.6. Statistical Analysis*

In the study on N limitation under elevated CO2, individual seedlings across the two chambers were used as the sample unit (*n* = 4–6). Two-way Analysis of Variance (ANOVA) (N × CO2) was used to test the di fferences in the treatment means of An, JPSII, JNPQ, JPSII+JNPQ, JNO and JChl. In the study on drought under elevated CO2, statistics are based on the individual plot (CO2 × water regime) in each chamber as the sample unit (*n* = 3). Three to six plants were measured in each plot.

A mean value from these plants was used as the estimate for that sample unit. Two-way ANOVA, with one between-subjects factor (CO2) and one within-subject factor (water regime), was used to test treatment di fferences in An, JPSII, JNPQ, JPSII + JNPQ, JNO and JChl. In the study on elevated O3 under elevated CO2, all statistics were based on the mean value of the individual plot (CO2 × O3 regime) as the sample unit (*n* = 3). These values were then averaged to provide the sample estimate for that replicate. Three-way ANOVA, with two between-subjects factors (CO2 and O3) and one within-subject factor (species), was used to test the differences in An, JPSII, JNPQ, JPSII + JNPQ, JNO and JChl, and leaf N.

#### **3. Results**

#### *3.1. Nitrogen Limitation under Elevated CO2*

When compared at the growth CO2, i.e., 360 μmol mol−<sup>1</sup> for the ambient-CO2-grown plants, and 720 μmol mol−<sup>1</sup> for the elevated-CO2-grown plants, higher An was observed in plants grown under elevated CO2 than in ambient-CO2 plants with adequate N supply, whereas no enhancement in An under elevated CO2 was observed with a limited N supply (Figure 1, Table 1). Conversely, no enhancement in JPSII was observed in plants grown under elevated CO2 with an adequate N supply, whereas the limited N supply resulted in lower JPSII irrespective of CO2 treatments. JNPQ was significantly higher in plants grown under limited N supply than those under adequate N supply. The sum of JNPQ + JPSII was not significantly different among the treatment combinations. As ABS was lower in the plants grown with limited N supply, higher JChl was observed in those plants. As a consequence of the increased JChl in addition to JNPQ, lower JNO was observed in the plants grown with limited N supply, in spite of significantly lower JPSII, irrespective of CO2 treatment.

**Figure 1.** Fate of light energy partitioning in the seedlings of Japanese white birch grown with N limitation under elevated CO2. JChl, JNPQ, JPSII and JNO were measured in the seedlings of Japanese white birch grown under ambient (*A*-CO2) and elevated CO2 (*E*-CO2) with adequate (+N) and limited N supply (-N). Open diamonds indicate net photosynthetic rate (An). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 1 month) under respective growth CO2 concentrations (i.e., 360 μmol mol−<sup>1</sup> for ambient-CO2-grown plants, and 720 μmol mol−<sup>1</sup> for elevated-CO2-grown plants) at saturating light (1200 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1). Values are means ± se (*n* = 4–6). Data were obtained from Kitao et al. [32]. JChl, JNPQ and JNO were newly calculated based on data from the previous study.


**Table 1.** *F* values of Analysis of Variance (ANOVA) to test the effects of various environmental stresses (N limitation, drought and elevated O3) under ambient or elevated CO2 on JNO, JPSII, JNPQ, JPSII+JNPQ, JChl, and An measured at respective growth CO2 concentrations. Significant effects are indicated in the table by \*\*\*: *p* ≤ 0.001, \*\*: *p* ≤ 0.01, \*: *p* ≤ 0.05, and ns: non-significant. Data were obtained from Kitao et al. [32–34].

#### *3.2. Drought under Elevated CO2*

Measurements of gas exchange and chlorophyll fluorescence were conducted at the growth CO2 (i.e., 360 μmol mol−<sup>1</sup> for the ambient-CO2-grown plants and 720 μmol mol−<sup>1</sup> for the elevated-CO2-grown plants) when soils were most dried on the previous day of irrigation (i.e., just prior to the scheduled watering). Intercellular CO2 concentration (Ci) was higher under elevated CO2, but lower under drought (Figure 2). Irrespective of the large variation of Ci itself, JChl, JNPQ, JNO and JPSII+JNPQ were not significantly different among the treatment combinations (Figure 2, Table 1). Only JPSII was significantly lower in the plants grown under elevated CO2, whereas no significant difference in An was observed among the treatment combinations.

**Figure 2.** Fate of light energy partitioning with drought under elevated CO2. Data are plotted as a function of intercellular CO2 concentration. JChl, JNPQ, JPSII and JNO were measured in the seedlings

of Japanese white birch grown under ambient and elevated CO2 with adequate (daily) and limited (once-weekly) water supply. AD: Ambient CO2 + once-weekly irrigation; AW: Ambient CO2 + daily-irrigation; ED: Elevated CO2 + once-weekly irrigation; EW: Elevated CO2 + daily-irrigation. Open diamonds indicate net photosynthetic rate (An). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 1 month) under the most dried conditions under respective growth CO2 concentrations (i.e., 360 μmol mol−<sup>1</sup> for ambient-CO2-grown plants, and 720 μmol mol−<sup>1</sup> for elevated-CO2-grown plants) at saturating light (1000 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1). Values are means ± se (*n* = 3). Data were obtained from Kitao et al. [33]. JChl, JNPQ and JNO were newly calculated based on data from the previous study.

#### *3.3. Elevated O3 under Elevated CO2*

An measured at the respective growth CO2 (i.e., 380 μmol mol−<sup>1</sup> for the ambient-CO2-grown plants and 550 μmol mol−<sup>1</sup> for the elevated-CO2-grown plants) increased under elevated CO2, but decreased under elevated O3 (Figure 3, Table 1). An was significantly different between *Q. mongolica* and *Q. serrata*, and the effects of CO2 and O3 were also different between species (Table 1). JPSII increased under elevated CO2, but decreased under elevated O3, whereas JNPQ decreased under elevated CO2 but increased under elevated O3. As a result, no significant differences were observed in JPSII+JNPQ among the treatment combinations or across species. JChl was neither affected by CO2, O3 nor species. Significantly lower JNO was observed in the plants grown under elevated O3. Area-based leaf N content (Narea) was not significantly different among the treatment combinations, whereas significantly higher Narea was observed in *Q. serrata* (Figure 4, Table 2).

**Figure 3.** Fate of light energy partitioning under elevated O3 and CO2. Here, JChl, JNPQ, JPSII and JNO were measured in the seedlings of Japanese oak (*Q. mongolica*) and Konara oak (*Q. serrata*) grown

under ambient (*A*-O3) and elevated O3 (*E*-O3), combined with ambient (*A*-CO2) and elevated CO2 (*E*-CO2). Open diamonds indicate the net photosynthetic rate (An). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 2 months) under respective growth CO2 concentrations (i.e., 380 μmol mol−<sup>1</sup> for ambient-CO2-grown plants, and 550 μmol mol−<sup>1</sup> for elevated-CO2-grown plants) at saturating light (1500 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1). Values are means ± se (*n* = 3). An was obtained from Kitao et al. [34].

**Figure 4.** Area-based leaf N content (Narae) in the seedlings of Japanese oak (*Q. mongolica*) and Konara oak (*Q. serrata*) grown under ambient (*A*-O3) and elevated O3 (*E*-O3), combined with ambient (*A*-CO2) and elevated CO2 (*E*-CO2). Values are means ± se (*n* = 3).

**Table 2.** *F* values of three-way ANOVA with two between-subjects factors (CO2 and O3) and one within-subject factor (species), to test the effects of CO2 (*F*1,8), O3 (*F*1,8), species (*F*1,8), CO2 × O3 (*F*1,8), CO2 × Species (*F*1,8), O3 × Species (*F*1,8), and CO2 × O3 × Species (*F*1,8) on area-based leaf nitrogen content (Narea). The symbols ns and \* denote non-significant (*p* > 0.05) and significant (*p* ≤ 0.05) effects, respectively.


#### **4. Discussion**

#### *4.1. Nitrogen Limitation under Elevated CO2*

Nitrogen plays a key role in photosynthesis, since Rubisco, a key-enzyme of photosynthesis, is the largest sink of N in a leaf [22], and also a considerable amount of N is involved in proteins related to linear electron transport [23,37]. Plants grown under elevated CO2 often show photosynthetic acclimation typically accompanied with a decrease in the maximum capacity of Rubisco carboxylation, known as photosynthetic down-regulation, particularly under limited nitrogen availability [27,35]. When Japanese white birch seedlings were grown under the combinations of CO2 and N treatments, leaves showed higher Narea with higher N supply, but lower Narea under elevated CO2 treatment [32]. In the present study, elevated CO2 had no effect on JNO, whereas limited N decreased JNO, suggesting a lower risk of photodamage under N limitation, irrespective of lower An [5]. The decreases in electron transport rate (JPSII) by N limitation and photosynthetic down-regulation under elevated CO2 were fully-compensated by regulated thermal energy dissipation (JNPQ), since the sum of JPSII and JNPQ was not significantly different across the treatment combinations. Conversely, the decrease in JNO under limited N resulted mainly from the increased loss of absorbed light energy, indicated by the increase in JChl.

#### *4.2. Drought under Elevated CO2*

Drought-induced stomatal closure leads to low intercellular CO2 (Ci) [1]. Leaves developed under long-term drought display higher photosynthetic capacity, accompanied with higher Narea, thus compensating the reduced photosynthetic performance under low Ci [38–40]. In the present study, seedlings of Japanese white birch were grown under elevated CO2 and long-term drought with limited N supply. Photosynthetic capacity, indicated by the maximum rate of Rubisco carboxylation (Vc,max), was previously shown to increase by long-term drought accompanied with higher Narea, whereas elevated CO2 decreased Vc,max with lower Narea [33]. In combination of changes in Vc,max with different Ci, An was not significantly different among the treatment combinations. In spite of similar An, JPSII decreased under elevated CO2, maybe because of a suppression of photorespiration under elevated CO2 (720 μmol mol−1) [41]. The decrease in JPSII under elevated CO2 was well compensated by a regulated photoprotective reaction (JNPQ) [2,5], leading to unchanged JNO under the combinations of CO2 and water treatments. An increase in Y(NO) was reported in mature leaves of *A. thaliana* under water deficit by withholding water, whereas a less extent of increase in Y(NO) was observed in young leaves, suggesting higher acclimating capacity, preventing oxidative damage in younger leaves [17]. In the present study, as the leaves of Japanese birch seedlings had flushed and developed during the relatively moderate drought treatment, they might fully acclimate to long-term drought, preventing photodamage [38–40].

#### *4.3. Elevated O3 under Elevated CO2*

Tropospheric ozone (O3) levels continue to increase globally [42,43], concurrently occurring with an increase in atmospheric CO2 concentration [44]. Contrary to elevated CO2, which may enhance plant growth in the short term [45,46], elevated O3 generally reduces plant growth via a reduction in photosynthetic rate and increased respiration rate [30,47]. Deciduous broadleaf trees native to Japan, Japanese oak (*Quercus mongolica*) and Konara oak (*Q. serrata*), were exposed to free air enriched with elevated O3 (twice ambient O3) and/or CO2 (550 μmol mol−<sup>1</sup> as target). An in the fully-expanded second-flushed leaves, measured at each growth CO2, reduced by elevated O3 but enhanced by elevated CO2, irrespective of species. As An was enhanced under elevated CO2 with no difference in Narea among the treatment combinations, photosynthetic down-regulation, which is often induced by elevated CO2 under limited N availability [32,35], was not apparent in the present study of a free-air CO2 and O3 exposure without limitations of root growth [34]. Furthermore, reduced leaf N, accompanied with a reduction in An under elevated O3 [48], was not observed in the present study, suggesting that causes other than leaf N reduction might be predominant to decrease An, such as an oxidative stress in the chloroplast [49]. JPSII was also reduced by elevated O3, but increased by elevated CO2, as well as An. In contrast, JNPQ was increased by elevated O3, but decreased by elevated CO2, which might fully compensate the changes in JPSII, as indicated by the constant JPSII+JNPQ. It is noteworthy that JNO decreased under elevated O3, which means that elevated O3 would not necessarily increase the risk of photodamage in these species.

#### *4.4. Regulated and Non-regulated Non-photochemical Quenching under Elevated CO2*

In the present study, we investigated the fate of light energy absorbed by a leaf under various environmental stresses combined with elevated CO2. We particularly focused on JNO, a measure of constitutive, non-regulated, non-photochemical energy dissipation, because an increase in JNO suggests an increase in the risk of photodamage [2,5]. As a whole, photoprotective thermal energy dissipation indicated by JNPQ may well compensate for the decreases in JPSII under environmental stresses, since JPSII+JNPQ was rather constant throughout the various stresses, even under elevated CO2. If plants can keep JPSII constant, there is a high potential for preventing the accumulation of excess energy [25,38–40]. However, if JPSII is restricted under limited N supply or by other environmental stress such as elevated O3, xanthophyll-related regulated thermal energy dissipation (JNPQ) would act as an efficient safety valve, which does not need N investment [8].

Furthermore, although drought and elevated CO2 had no effects on JNO, N limitation and elevated O3 resulted in decreases in JNO, in contrast to expected stress responses (i.e., increases in JNO), which can be considered as an *adaptive response* in the framework of pre-conditioning to cope with further environmental stresses [50,51]. By doing so, JNO may be decreased to such an extent that will offset high increases that would occur under further stress. This novel mechanism builds upon an extended body of literature showing the biological capacity of a variety of organisms to display hormetic *adaptive responses* which eventually act as biological shields against following health threats [50–52]. Such *adaptive responses* for coping with stress are activated by low/mild severity of stress, at levels that are (often far) lower than the level beyond which toxicological, adverse responses occur [50–52]. This suggests that NPQ can compensate for the effects of following more severe environmental stress, but if the stress is too severe (e.g., acute exposure), increased NPQ may not be enough to fully compensate for the negative effects of stress.

Whereas it was difficult to explicitly determine the factor inducing lower JNO under elevated O3 (maybe the integrated effects of JNPQ + JChl), an increase in JChl apparently contributed to reducing JNO under limited N. Thus, in addition to the fractions of absorbed light energy partitioning, based on chlorophyll fluorescence (Y(II), Y(NPQ) and Y(NO)), reduced chlorophyll pigments should be taken into account as a photoprotective reaction for assessing environmental stresses by using chlorophyll fluorescence measurements [53].

Similar to the present study, a stable or even lower Y(NO) due to the decline in Y(II), accompanied with the increase in Y(NPQ), was also reported in paraquat-exposed *Arabidopsis thaliana* [11,18] and in Al-exposed *A. thaliana* [12]. The decrease in JNO may denote also decreased ROS production [17]. Non-regulated, non-photochemical quenching consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of singlet oxygen (1O2) via the triplet state of chlorophyll (3chl\*) [10,11,13]. Since JNO declined, it seems that JNPQ was sufficient enough to protect plants from ROS, by exhibiting lower 1O2 production, and preventing the photosynthetic apparatus from oxidative damage [12].

#### **5. Conclusions**

Based on the results from three deciduous broadleaf tree species in the present study, even when photosynthesis and JPSII were reduced by environmental stresses, photoprotective mechanisms including JNPQ and JChl could suppress the rise of JNO in the leaves developed under the stresses, consequently preventing photodamage even under future-coming elevated CO2 conditions.

**Author Contributions:** M.K., and H.T. designed the study. M.K., H.T., S.K., H.H., K.Y. and M.K. collected the photosynthetic data, performed the analysis, and hence equally contributed to this study. M.K. led the writing with input from E.A. and T.K. All authors also discussed the results and commented on the manuscript.

**Funding:** This study was supported in part by JSPS KAKENHI Grant Number JP17K19301, JP17F17102 and JP17H03839. Evgenios Agathokleous was a JSPS International Research Fellow (ID No: P17102).

**Acknowledgments:** We thank K Mima and K Sakai for leaf N analyses, and express our sincere appreciation to V. Hurry for his helpful suggestions on our article. E.A. acknowledges multi-year support from The Startup Foundation for Introducing Talent of Nanjing University of Information Science & Technology (NUIST), Nanjing, China.

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