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

: 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 O 3 in Japanese oak ( Quercus mongolica var. crispula ) and Konara oak ( Q. serrata ) under future-coming elevated CO 2 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 (J NO ) were observed in plants grown under N limitation, drought and elevated O 3 in ambient or elevated CO 2 . This suggests that the risk of photodamage caused by excessive light energy was not increased by environmental stresses reducing photosynthesis, irrespective of CO 2 concentrations. The rate of regulated non-photochemical quenching (J NPQ ), which contributes to regulating photoprotective thermal dissipation, could well compensate decreases in the photosynthetic electron transport rate through photosystem II (J PSII ) under various environmental stresses, since J NPQ + J PSII was constant across the treatment combinations. It is noteworthy that even decreases in J NO were observed under N limitation and elevated O 3 , irrespective of CO 2 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 ﬀ ects of lethal stress, but may be critical for coping with non-lethal stress and regulating homeostasis. Regarding the three deciduous broadleaf tree species, elevated CO 2 appears not to inﬂuence the plant responses to environmental stresses from the viewpoint of susceptibility to photodamage.


Introduction
Although light is essential for plant growth, plants can suffer from excessive light, especially when combined with other environmental stresses. Light energy absorbed by a leaf is mainly consumed and non-regulated non-photochemical quenchings were newly calculated based on data from the previous studies [32,33]. Conversely, regarding "elevated O 3 under elevated CO 2 ", all data except for A n were not published previously (cf. [34]).

N Limitation under Elevated CO 2
Data of Japanese white birch (Betula platyphylla var. japonica) seedlings grown under limited N and elevated CO 2 were obtained from the study by Kitao et al. [32]. Experiments of N limitation under elevated CO 2 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 CO 2 treatments: 360 µmol mol −1 (ambient CO 2 treatment, A-CO 2 ); and 720 µmol mol −1 (elevated CO 2 treatment, E-CO 2 ) 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 −1 week −1 for 7 weeks during CO 2 treatment, whereas the latter one was conducted as 100 mg N pot −1 only once at the onset of CO 2 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 (N area ) 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].

Drought under Elevated CO 2
Data of Japanese white birch seedlings grown under limited water supply and elevated CO 2 were obtained from the study by Kitao et al. [33]. Experiments of drought under elevated CO 2 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 CO 2 treatments i.e., 360 (ambient CO 2 treatment: A-CO 2 ) and 720 µmol mol -1 (elevated CO 2 treatment: E-CO 2 ) 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-CO 2 + adequate water supply: −0.13, A-CO 2 + drought: −0.52, E-CO 2 + adequate water supply: −0.12 and E-CO 2 + 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.
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 CO 2 (Target set, 550 µmol mol −1 ), elevated O 3 (Target set, twice-ambient), and elevated CO 2 + O 3 (550 µmol mol −1 CO 2 and twice-ambient O 3 ). Plants were grown under the treatments for two growing seasons. Measurements of gas exchange and chlorophyll fluorescence were conducted in the second growing season.

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, Effeltrich, Germany) for plants grown under "N limitation with CO 2 enrichment", or a leaf chamber fluorometer (Li-6400-40, Li-Cor) for plants grown under "drought, and elevated O 3 under elevated CO 2 ". Details are described in Kitao et al. [32][33][34]. The net photosynthetic rate (A n ), 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][3][4][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/F m , and Y(NPQ) = F/F m ' − F/F m , 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 O 3 under elevated CO 2 , we measured F, F m ' and F o ' (the minimum fluorescence yield during illumination) during the gas exchange measurements, but did not measure F m . We measured F v /F m 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 O 3 -treated plants [34] ). 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][33][34]. Based on the chlorophyll fluorescence parameters, the electron transport rate (J PSII ) was calculated as J PSII = Y(II) × ABS × light intensity × 0.5 [6]. Analogous to J PSII , the rate of regulatory thermal dissipation (J NPQ ) and the rate of non-regulatory energy dissipation via heat or fluorescence (J NO ) 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 (J Chl ) was estimated as J Chl = (1 − ABS) × light intensity × 0.5.

Leaf N Content
Regarding 'elevated O 3 under elevated CO 2 ', the leaves were sampled after the measurements and used for a determination of N area 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).

Statistical Analysis
In the study on N limitation under elevated CO 2 , individual seedlings across the two chambers were used as the sample unit (n = 4-6). Two-way Analysis of Variance (ANOVA) (N × CO 2 ) was used to test the differences in the treatment means of A n , J PSII , J NPQ , J PSII +J NPQ , J NO and J Chl . In the study on drought under elevated CO 2 , statistics are based on the individual plot (CO 2 × 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 (CO 2 ) and one within-subject factor (water regime), was used to test treatment differences in A n , J PSII , J NPQ , J PSII + J NPQ , J NO and J Chl . In the study on elevated O 3 under elevated CO 2 , all statistics were based on the mean value of the individual plot (CO 2 × O 3 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 (CO 2 and O 3 ) and one within-subject factor (species), was used to test the differences in A n , J PSII , J NPQ , J PSII + J NPQ , J NO and J Chl , and leaf N.

Nitrogen Limitation under Elevated CO 2
When compared at the growth CO 2 , i.e., 360 µmol mol −1 for the ambient-CO 2 -grown plants, and 720 µmol mol −1 for the elevated-CO 2 -grown plants, higher A n was observed in plants grown under elevated CO 2 than in ambient-CO 2 plants with adequate N supply, whereas no enhancement in A n under elevated CO 2 was observed with a limited N supply ( Figure 1, Table 1). Conversely, no enhancement in J PSII was observed in plants grown under elevated CO 2 with an adequate N supply, whereas the limited N supply resulted in lower J PSII irrespective of CO 2 treatments. J NPQ was significantly higher in plants grown under limited N supply than those under adequate N supply. The sum of J NPQ + J PSII was not significantly different among the treatment combinations. As ABS was lower in the plants grown with limited N supply, higher J Chl was observed in those plants. As a consequence of the increased J Chl in addition to J NPQ , lower J NO was observed in the plants grown with limited N supply, in spite of significantly lower J PSII , irrespective of CO 2 treatment. Open diamonds indicate net photosynthetic rate (A n ). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 1 month) under respective growth CO 2 concentrations (i.e., 360 µmol mol −1 for ambient-CO 2 -grown plants, and 720 µmol mol −1 for elevated-CO 2 -grown plants) at saturating light (1200 µmol m −2 s −1 ). Values are means ± se (n = 4-6). Data were obtained from Kitao et al. [32]. J Chl , J NPQ and J NO were newly calculated based on data from the previous study. Measurements of gas exchange and chlorophyll fluorescence were conducted at the growth CO 2 (i.e., 360 µmol mol −1 for the ambient-CO 2 -grown plants and 720 µmol mol −1 for the elevated-CO 2 -grown plants) when soils were most dried on the previous day of irrigation (i.e., just prior to the scheduled watering). Intercellular CO 2 concentration (C i ) was higher under elevated CO 2 , but lower under drought ( Figure 2). Irrespective of the large variation of C i itself, J Chl , J NPQ , J NO and J PSII +J NPQ were not significantly different among the treatment combinations ( Figure 2, Table 1). Only J PSII was significantly lower in the plants grown under elevated CO 2 , whereas no significant difference in A n was observed among the treatment combinations. Data are plotted as a function of intercellular CO 2 concentration. J Chl , J NPQ , J PSII and J NO were measured in the seedlings of Japanese white birch grown under ambient and elevated CO 2 with adequate (daily) and limited (once-weekly) water supply. AD: Ambient CO 2 + once-weekly irrigation; AW: Ambient CO 2 + daily-irrigation; ED: Elevated CO 2 + once-weekly irrigation; EW: Elevated CO 2 + daily-irrigation. Open diamonds indicate net photosynthetic rate (A n ). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 1 month) under the most dried conditions under respective growth CO 2 concentrations (i.e., 360 µmol mol −1 for ambient-CO 2 -grown plants, and 720 µmol mol −1 for elevated-CO 2 -grown plants) at saturating light (1000 µmol m −2 s −1 ). Values are means ± se (n = 3). Data were obtained from Kitao et al. [33]. J Chl , J NPQ and J NO were newly calculated based on data from the previous study.

Elevated O 3 under Elevated CO 2
A n measured at the respective growth CO 2 (i.e., 380 µmol mol −1 for the ambient-CO 2 -grown plants and 550 µmol mol −1 for the elevated-CO 2 -grown plants) increased under elevated CO 2 , but decreased under elevated O 3 (Figure 3, Table 1). A n was significantly different between Q. mongolica and Q. serrata, and the effects of CO 2 and O 3 were also different between species (Table 1). J PSII increased under elevated CO 2 , but decreased under elevated O 3 , whereas J NPQ decreased under elevated CO 2 but increased under elevated O 3 . As a result, no significant differences were observed in J PSII +J NPQ among the treatment combinations or across species. J Chl was neither affected by CO 2 , O 3 nor species. Significantly lower J NO was observed in the plants grown under elevated O 3 . Area-based leaf N content (N area ) was not significantly different among the treatment combinations, whereas significantly higher N area was observed in Q. serrata (Figure 4, Table 2). , combined with ambient (A-CO 2 ) and elevated CO 2 (E-CO 2 ). Open diamonds indicate the net photosynthetic rate (A n ). Measurements were conducted for fully-developed mature leaves (leaf age was approx. 2 months) under respective growth CO 2 concentrations (i.e., 380 µmol mol −1 for ambient-CO 2 -grown plants, and 550 µmol mol −1 for elevated-CO 2 -grown plants) at saturating light (1500 µmol m −2 s −1 ). Values are means ± se (n = 3). A n was obtained from Kitao et al. [34].

Nitrogen Limitation under Elevated CO 2
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 CO 2 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 CO 2 and N treatments, leaves showed higher N area with higher N supply, but lower N area under elevated CO 2 treatment [32]. In the present study, elevated CO 2 had no effect on J NO , whereas limited N decreased J NO , suggesting a lower risk of photodamage under N limitation, irrespective of lower A n [5]. The decreases in electron transport rate (J PSII ) by N limitation and photosynthetic down-regulation under elevated CO 2 were fully-compensated by regulated thermal energy dissipation (J NPQ ), since the sum of J PSII and J NPQ was not significantly different across the treatment combinations. Conversely, the decrease in J NO under limited N resulted mainly from the increased loss of absorbed light energy, indicated by the increase in J Chl .

Drought under Elevated CO 2
Drought-induced stomatal closure leads to low intercellular CO 2 (C i ) [1]. Leaves developed under long-term drought display higher photosynthetic capacity, accompanied with higher N area , thus compensating the reduced photosynthetic performance under low C i [38][39][40]. In the present study, seedlings of Japanese white birch were grown under elevated CO 2 and long-term drought with limited N supply. Photosynthetic capacity, indicated by the maximum rate of Rubisco carboxylation (V c,max ), was previously shown to increase by long-term drought accompanied with higher N area , whereas elevated CO 2 decreased V c,max with lower N area [33]. In combination of changes in V c,max with different C i , A n was not significantly different among the treatment combinations. In spite of similar A n , J PSII decreased under elevated CO 2 , maybe because of a suppression of photorespiration under elevated CO 2 (720 µmol mol −1 ) [41]. The decrease in J PSII under elevated CO 2 was well compensated by a regulated photoprotective reaction (J NPQ ) [2,5], leading to unchanged J NO under the combinations of CO 2 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][39][40].

Elevated O 3 under Elevated CO 2
Tropospheric ozone (O 3 ) levels continue to increase globally [42,43], concurrently occurring with an increase in atmospheric CO 2 concentration [44]. Contrary to elevated CO 2 , which may enhance plant growth in the short term [45,46], elevated O 3 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 O 3 (twice ambient O 3 ) and/or CO 2 (550 µmol mol −1 as target). A n in the fully-expanded second-flushed leaves, measured at each growth CO 2 , reduced by elevated O 3 but enhanced by elevated CO 2 , irrespective of species. As A n was enhanced under elevated CO 2 with no difference in N area among the treatment combinations, photosynthetic down-regulation, which is often induced by elevated CO 2 under limited N availability [32,35], was not apparent in the present study of a free-air CO 2 and O 3 exposure without limitations of root growth [34]. Furthermore, reduced leaf N, accompanied with a reduction in A n under elevated O 3 [48], was not observed in the present study, suggesting that causes other than leaf N reduction might be predominant to decrease A n , such as an oxidative stress in the chloroplast [49]. J PSII was also reduced by elevated O 3 , but increased by elevated CO 2 , as well as A n . In contrast, J NPQ was increased by elevated O 3 , but decreased by elevated CO 2 , which might fully compensate the changes in J PSII , as indicated by the constant J PSII +J NPQ . It is noteworthy that J NO decreased under elevated O 3 , which means that elevated O 3 would not necessarily increase the risk of photodamage in these species.

Regulated and Non-regulated Non-photochemical Quenching under Elevated CO 2
In the present study, we investigated the fate of light energy absorbed by a leaf under various environmental stresses combined with elevated CO 2 . We particularly focused on J NO , a measure of constitutive, non-regulated, non-photochemical energy dissipation, because an increase in J NO suggests an increase in the risk of photodamage [2,5]. As a whole, photoprotective thermal energy dissipation indicated by J NPQ may well compensate for the decreases in J PSII under environmental stresses, since J PSII +J NPQ was rather constant throughout the various stresses, even under elevated CO 2 . If plants can keep J PSII constant, there is a high potential for preventing the accumulation of excess energy [25,[38][39][40]. However, if J PSII is restricted under limited N supply or by other environmental stress such as elevated O 3 , xanthophyll-related regulated thermal energy dissipation (J NPQ ) would act as an efficient safety valve, which does not need N investment [8].
Furthermore, although drought and elevated CO 2 had no effects on J NO , N limitation and elevated O 3 resulted in decreases in J NO , in contrast to expected stress responses (i.e., increases in J NO ), 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, J NO 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][51][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][51][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 J NO under elevated O 3 (maybe the integrated effects of J NPQ + J Chl ), an increase in J Chl apparently contributed to reducing J NO 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 J NO 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 ( 1 O 2 ) via the triplet state of chlorophyll ( 3 chl*) [10,11,13]. Since J NO declined, it seems that J NPQ was sufficient enough to protect plants from ROS, by exhibiting lower 1 O 2 production, and preventing the photosynthetic apparatus from oxidative damage [12].

Conclusions
Based on the results from three deciduous broadleaf tree species in the present study, even when photosynthesis and J PSII were reduced by environmental stresses, photoprotective mechanisms including J NPQ and J Chl could suppress the rise of J NO in the leaves developed under the stresses, consequently preventing photodamage even under future-coming elevated CO 2 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.