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Article

Effects of Combined CO2 and O3 Exposures on Net CO2 Assimilation and Biomass Allocation in Seedlings of the Late-Successional Fagus Crenata

by
Hiroyuki Tobita
1,*,
Masabumi Komatsu
1,
Hisanori Harayama
2,
Kenichi Yazaki
1,
Satoshi Kitaoka
1 and
Mitsutoshi Kitao
2
1
Department of Plant Ecology, Forestry and Forest Products Research Institute, Matsunosato 1, Tsukuba 305-8687, Japan
2
Hokkaido Research Center, Forestry and Forest Products Research Institute, Hitsujigaoka 7, Sapporo 062-8516, Japan
*
Author to whom correspondence should be addressed.
Climate 2019, 7(10), 117; https://0-doi-org.brum.beds.ac.uk/10.3390/cli7100117
Submission received: 15 July 2019 / Revised: 21 September 2019 / Accepted: 21 September 2019 / Published: 26 September 2019
(This article belongs to the Special Issue Air Pollution and Plant Ecosystems)
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Abstract

:
We examined the effects of elevated CO2 and elevated O3 concentrations on net CO2 assimilation and growth of Fagus crenata in a screen-aided free-air concentration-enrichment (FACE) system. Seedlings were exposed to ambient air (control), elevated CO2 (550 µmol mol−1 CO2, +CO2), elevated O3 (double the control, +O3), and the combination of elevated CO2 and O3 (+CO2+O3) for two growing seasons. The responses in light-saturated net CO2 assimilation rates per leaf area (Agrowth-CO2) at each ambient CO2 concentration to the elevated CO2 and/or O3 treatments varied widely with leaf age. In older leaves, Agrowth-CO2 was lower in the presence of +O3 than in untreated controls, but +CO2+O3 treatment had no effect on Agrowth-CO2 compared with the +CO2 treatment. Total plant biomass increased under conditions of elevated CO2 and was largest in the +CO2+O3 treatment. Biomass allocation to roots decreased with elevated CO2 and with elevated O3. Elongation of second-flush shoots also increased in the presence of elevated CO2 and was largest in the +CO2+O3 treatment. Collectively, these results suggest that conditions of elevated CO2 and O3 contribute to enhanced plant growth; reflecting changes in biomass allocation and mitigation of the negative impacts of O3 on net CO2 assimilation.

1. Introduction

Atmospheric carbon dioxide (CO2) concentrations are predicted to double during the next century, and tropospheric ozone (O3) levels have also continued to rise globally since pre-industrial times [1,2,3,4,5]. Both CO2 and O3 are recognized as anthropogenic air pollutants with opposing impacts on plant growth [6,7,8,9]. Elevated CO2 stimulates plant growth by enhancing photosynthetic carbon assimilation [10,11,12], although long-term exposure to elevated CO2 results in photosynthetic down-regulation, especially under limiting nutrient conditions [13,14]. Elevated O3 concentrations reduce net CO2 assimilation (A) and accelerate leaf senescence [15,16], potentially resulting in substantial losses of the C-sink strengths in trees [9,17,18,19,20].
Elevated CO2 may alleviate the negative effects of O3 on photosynthetic activity and plant growth [8,21]. Free-air concentration enrichment (FACE) experiments for CO2 and other ambient air experiments indicate a nonlinear interaction between plant responses to CO2 and O3 [7,22,23]. Hence, O3 exposure may alter the C metabolism and decrease C stocks for tree species, likely through changes in quantities or the compositions of nonstructural carbohydrates [24]. They may in turn alter C allocation in plants [19,25]. Increased O3 concentrations have been shown to shift carbon allocation in favor of aboveground plant tissues, especially leaves, at the expense of root growth, presumably to allocate resources for detoxification and repair of damaged leaves [6,16,26,27,28,29,30]. As elevated CO2 can alleviate the O3-induced reduction in A, shifts in biomass allocation into leaves may have an over-compensating effect on plant growth under the combination of elevated CO2 and O3. Although mechanisms of O3-induced responses in plants have been revealed [31], responses to elevated O3 vary widely among species [3,32]. Moreover, interactions between environmental variables such as elevated CO2, drought, and high temperature, and their effects on crop productivity, are poorly understood [31].
Scaling up from the laboratory to the field with FACE technologies will be crucial to the understanding of plant responses to elevated O3 and CO2 [31]. Yet free-air exposure experiments with combinations of elevated CO2 and O3 [33] and with elevated O3 only [17,34,35] are fewer than those performed in free air CO2 experiments [36,37,38,39], especially in Asia [40]. In Japan, free-air CO2 and O3 exposure experiments have been conducted under field conditions [41,42,43,44]. Kitao et al., [45] reported the growth responses to elevated CO2 and O3 in two Quercus species, Q. mongolica and Q. serrata, which have relatively high tolerance to O3 among Japanese tree species [46,47]. A significant enhancement of growth was observed in the seedlings grown under the combination of elevated CO2 and O3, compared with those grown under elevated CO2 alone. This growth enhancement was considered as an over-compensating response, consisting of two processes: (1) preferential biomass allocation into leaves induced by elevated O3, and (2) enhanced A by elevated CO2.
Fagus crenata is a representative deciduous broadleaf tree species, widely distributing in the cool temperate forests in Japan [48]. This species is more susceptible to O3 than the Quercus species described above [45]. Even at the current ambient O3 levels, higher O3 uptake by a forest of F. crenata resulted in an accelerated autumn senescence based on the observations by a flux tower [49]. Previous studies of plant-level F. crenata responses to interactions of CO2 and O3 produced contradictory results, with one study showing increased growth by combined elevated CO2 and O3 in greenhouse experiments [50], and another showing that elevated CO2 concentrations did not mitigate the negative effects of elevated O3 on plant growth [51]. Whether elevated CO2 concentrations ameliorate the negative impacts of O3 on growth is not certain, even in the same species [52]. To understand growth responses of F. crenata to elevated CO2 and O3, changes in C allocation into leaves and effects on A should be investigated collectively.
Quercus mongolica and Q. serrata, mid-successional species with a flush and succeeding-type of leaf emergence [53], can flush new shoots several times under ideal conditions. Unlike these Quercus species, F. crenata, a shade-tolerant late-successional species [48], has a flush type shoot developmental pattern [53], in which shoots are usually flushed only once in spring. We hypothesized that growth enhancement might be observed in the seedlings of F. crenata grown under the combination of elevated O3 and CO2, whereas the related growth enhancement is unlikely to be as large as those in the two Quercus species [45], because the leaf expansion pattern of F. crenata has lower plasticity to changes in environmental conditions. To test this hypothesis, we performed screen-aided FACE experiments and investigated the combined effects of elevated CO2 and O3 concentrations on A (as a leaf level trait) and biomass allocation (as a plant-level trait) in F. crenata seedlings without limiting root growth.

2. Material and Methods

2.1. Experimental Design

Screen-aided free-air concentration enrichment (FACE) experiments with elevated O3 and CO2 were performed in the facility used in previous studies [41,45]. The experimental field is located in the nursery of the Forestry and Forest Products Research Institute in Tsukuba, Japan (36°00′ N, 140°08′ E, 20 m a.s.l.). Plants were exposed to unchanged ambient air (control), elevated CO2 (+CO2; 550 µmol mol−1 CO2), elevated O3 (+O3; twice-ambient), or elevated CO2 and elevated O3 (+CO2+O3; 550 µmol mol−1 CO2 and twice-ambient O3). A total of twelve frames (2 m width × 2.5 m height) were installed with three replicates for each treatment. CO2 and O3 were supplied during the daytime with a proportional integral derivative (PID) control system comprised of digital controllers (Model SDC35, Azbil Corporation, Tokyo, Japan). Gaseous CO2 was obtained from liquid CO2 (AIR WATER INC., Osaka, Japan). O3 was supplied by an ozone generator (Model PZ2A; Kofloc, Kyoto, Japan). CO2 concentration was measured by two CO2 monitors (Carbon Dioxide Probe, Model GMP343; Vaisala, Helsinki, Finland); in addition, CO2 concentration in the frames was monitored by an infrared CO2 analyzer (Model LI-820; LI-COR Inc., Lincoln, NE, USA). Ozone concentration was measured by both an O3 analyzer (Model EG-3000F; Ebara Jitsugyo Co. Ltd., Kanagawa, Japan) and an O3 monitor (Model 205; 2B Technologies, Boulder, CO, USA). The exposure periods for CO2 and O3 extended from 20 May to 30 November in 2011, and from 25 April to 20 November in 2012 (these data are the same as those reported by Hiraoka et al. [41]). Mean (± SE) daytime (7:00–17:00) CO2 and O3 concentrations during treatments were 36.1 ± 1.0 and 65.3 ± 2.0 ppb in 2011 and 36.4 ± 0.5 and 61.5 ± 6.2 ppb in 2012 for ambient and elevated O3, respectively, and were 378 ± 2.5 and 562 ± 16.9 ppm in 2011 and 377 ± 2.9 and 546 ± 21.3 ppm in 2012 for ambient and elevated CO2, respectively. Frames were surrounded by two transparent windscreens (50 cm height), encircled at 15 and 75 cm above the soil. The windscreens facilitated turbulent mixing of CO2 and O3, which were injected with ambient air through holes in vertical polyethylene tubes (2 m in height at 20-cm intervals around the frames) [54]. During the growth seasons from the beginning of April to the end of October in 2011 and 2012, mean monthly temperatures ranged from 8.2 °C to 28.4 °C. Precipitation for the growth period was 1096 mm in 2011 and 1019 mm in 2012 [41].

2.2. Plant Materials

Fagus crenata is a representative deciduous broadleaf tree species in Japan and is mainly distributed in deciduous broadleaf forests of the warm-temperate zone of East Asia. Fagus crenata have late-successional traits. Their shoot developmental pattern is classified as flush type shoot growth [53], in which shoots are usually flushed once in the spring. Nine current-year, dormant seedlings of F. crenata (about 5 cm in height) were transplanted directly into the ground in each frame in March 2011. Tree seedlings were grown for two growth seasons under each of the treatment conditions described above. The maximum height of the seedlings at the end of this experiment was 132.7 cm, which was lower than the frame height.

2.3. Gas-Exchange Measurements

Light-saturated net CO2 assimilation rates per leaf area (Agrowth-CO2) in each ambient CO2 concentration level (at 380 µmol mol−1 and 550 µmol mol−1 for ambient and elevated CO2 conditions, respectively) were measured five times in first-flush leaves during mid-May, early July, early August, mid-September, and late October of the second year of CO2 and O3 exposures. Leaf photosynthetic parameters at ambient (380 µmol mol−1) and elevated CO2 (550 µmol mol−1) concentrations were measured in three individual seedlings per treatment combination using a portable photosynthesis system (Li-6400, Li-Cor) and a leaf chamber fluorometer (Li-6400-40, Li-Cor). Measurements were taken with the block temperature set at 27 °C and at a saturating light intensity of 1500 µmol m−2 s−1. Light was provided by a red/blue LED array with blue light comprising 10% of the total PFD. In preliminary studies, this light intensity was sufficient to saturate A in F. crenata. Gas-exchange measurements were conducted from 9:00 to 15:00. Prior to these measurements, sunscreens were placed over the frames to prevent direct sunlight from illuminating the leaves.
In August 2012, A-Ci relationships of the first flush leaves were determined and used to calculate maximum rates of carboxylation at 25 °C (Vcmax25). The A-Ci curves were determined in six steps with saturating light at 1500 µmol m−2 s−1, starting from an ambient CO2 concentration for each treatment (380 or 550) followed by 100-, 200-, 380-, 550-, and 1000-μmol CO2 mol−1. Measurements were taken at a block temperature of 27 °C. Maximum rates of carboxylation (Vcmax) were estimated using the following equation of the Farquhar-type model [55]: A = Vcmax (Cc − Γ*)/{Cc + Kc × (1 + Oi/Ko)} − Rd, where Cc is the CO2 concentration in the chloroplasts, Γ* is the CO2 compensation point, Kc and Ko are the Michaelis–Menten constants for carboxylation and oxygenation, Oi is the intercellular O2 concentration, and Rd is the rate of daytime respiration. In this study, we ignored possible CO2 diffusion limitations within leaves, and used Ci as a proxy for Cc. Values for the coefficients Kc, Ko, and Γ* were 404.9, 278.4, and 42.75 μmol mol−1, respectively, at 25 °C [56]. Vcmax was calculated by fitting the equation to the initial slope of the A/Ci curve (Ci < 300).

2.4. Chlorophyll Fluorescence Measurements

To quantitate the degree of aging-related senescence following CO2 and O3 treatments, we determined initial (F0) and maximum fluorescence (Fm) values in all leaves used for the gas exchange measurements after overnight dark-adaptation using a portable fluorometer (MINI-PAM, Walz, Effeltrich, Germany). Saturating pulse of 7000 mol m−2 s−1 for 1 s was used to determine Fm values. The ratio of variable to maximum fluorescence (Fv/Fm where Fv = Fm − F0) represents the maximum PS II photochemical efficiency [57].

2.5. Leaf Characteristics

Leaves were collected after the measurements of Fv/Fm and leaf areas were measured using a scanner and Image J software (NIH, Bethesda, MD, USA). Dry weights of each leaf were measured after oven-drying at 70 °C and leaf mass per leaf area (LMA) was calculated for each leaf. Nitrogen concentrations of each leaf (leaf Nmass) were analyzed using an NC-analyzer (NT-900, SUMIKA Chem., Osaka, Japan), and N contents per leaf area (leaf Narea) were calculated using LMA.

2.6. Growth Measurements

In November of the second year, gas-exchange measurements were completed and all seedlings were harvested (n = 15, control; n = 15, +CO2; n = 22, +O3; n = 21, +CO2+O3). Root systems were then excavated using an air excavation tool (Air Schop, KF Company, Sakai, Japan) to loosen soils [45]. Loosened soils were then carefully removed by hand using small stainless-steel rakes (Bonsai rake, Kikuwa, Sanjo, Niigata, Japan). Dry weights of leaves, current-year shoots, stems, and roots were determined after oven-drying to a constant weight at 70 °C.

2.7. Statistical Analysis

Significant effects of CO2 and O3 treatments on plant biomass, organ biomass (leaves, current-year shoots, stems and non-current-year shoots, and roots), second-flush shoot lengths, Agrowth-CO2, gsw-growth-CO2, Ci, leaf Nmass, leaf Narea, and LMA were identified using ANOVA with mean values from each frame (CO2 × O3 regimen; n = 12). Values were averaged to calculate sample estimates for each replicate. Standardized major axis tests and routines (SMA) regression [58,59] analyses were used to examine relationships among log10-transformed whole plant and plant tissue (g plant−1) biomasses using the SMATR 3.3 package [60]. Differences in elevations of SMA regressions were tested among treatments using Sidak’s adjusted pair-wise tests, depending on the significance of the null hypothesis that the slopes were equal. Analyses were performed using data from all plants of each treatment group. All statistical analyses were performed using R version 3.2.3 [61].

3. Results

3.1. Photosynthesis

Net CO2 assimilation rate responses to CO2 and O3 treatments varied widely with leaf age (Figure 1A, Table 1). Although Agrowth-CO2 increased following treatment with elevated CO2 concentrations in younger leaves (May, p = 0.088; July, P = 0.082), no positive effects of elevated CO2 were observed in older leaves (August to October). During August, Vcmax25 declined more in the +O3 treatment group than in control plants, yet no significant effects of CO2 and O3 treatments were identified (Table 1). In October, Agrowth-CO2 did not significantly decline due to elevated O3 exposure, and predawn Fv/Fm was lower in the +O3 treatment group than in all other groups (Table 1). Stomatal conductance (gsw-growth-CO2) decreased by elevated CO2 in May (p = 0.036) and July (p = 0.017) (Figure 1B, Table 1). A tendency for effects of the O3 were also detected only in July (p = 0.058). During September and October, seedlings of the +CO2+O3 group tended to have the lowest gsw-growth-CO2 values. Finally, elevated O3 had no effect on Ci throughout the growing season (Figure 1C).

3.2. Leaf Characteristics

Leaf Nmass of F. crenata seedlings was decreased by CO2 treatment in May (F1,8 = 25.8; p = 0.001), and August (F1,8 = 3.8; p = 0.088; Figure 2A). In August, O3 treatments also affected leaf Nmass (F1,8 = 9.2; p = 0.016). Effects of CO2 in May (F1,8 = 6.9; p = 0.031) and its interactions with O3 in July (F1,8 = 4.0; p = 0.080) on LMA were observed (Figure 2B). Specifically, +CO2+O3-treated seedlings had the highest LMA values among the four treatment groups in all growth periods, yet no significant effects of CO2 and O3 treatments were identified. Leaf Narea values showed interactive effects of CO2 and O3 treatments in September (F1,8 = 7.1; p = 0.028), May (F1,8 = 3.8; p = 0.087) and August (F1,8 = 3.7; p = 0.091) in addition to the effects of O3 treatments in August (Figure 2C; F1,8 = 21.5; p = 0.0017).

3.3. Plant Biomass

Elevated CO2 concentrations had positive effects on the whole plant biomasses (Total) of F. crenata seedlings (Table 2; p = 0.033). Seedlings of the +CO2+O3 treatment group tended to exhibit the largest average plant biomass among the four treatment groups (p = 0.037, in one-way ANOVA). Though no significant effects of CO2 and O3 treatments were identified in dry matter distribution, elevated CO2 concentrations showed a tendency to decrease RWR (p = 0.078) and increase aboveground biomass to root ratio (S:R ratio) (p = 0.066), and leaf to root ratio (L:R ratio) tended to increase by elevated O3 treatment (p = 0.065) (Table 3). All treatment groups included some seedlings that had a second flush leaf production. Yet only following +CO2+O3 treatments did all plants produce second-flush leaves (77.8% ± 14.7%, Control; 68.9% ± 17.4%, +CO2; 81.9% ± 10.8%, +O3; and 100%, +CO2+O3; these values indicated mean ± SE). Lengths of second-flush shoots (mean ± SE) were also affected by CO2 concentrations (p = 0.030) and were 31.4 ± 12.4 cm in control plants, 51.3 ± 5.9 cm in +CO2 treated plants; 32.7 ± 2.9 cm in +O3 treated plants, and 49.8 ± 1.1 cm in +CO2+O3 treated plants.

3.4. Biomass Allocation

In our allometric assessments of relationships between whole plant weights and root weights, regression slopes did not differ among treatments (p = 0.231; Figure 3A, Table 4). In case the common slopes of 1.015 (0.947–1.091; ±95% confidence intervals (CI)) were used, magnitudes of relationships differed significantly between treatment groups (p = 0.0002). Specifically, seedlings from the +CO2+O3 treatment group had smaller elevations than those of the control and the +O3 treatment groups. Allometric assessments of root and leaf weights also gave slopes that did not differ between treatments (p = 0.114; Figure 3B, Table 4). At common slopes of 0.979 (0.831–1.153; ± 95% CI), magnitudes of these relationships differed significantly between treatment groups (p = 0.0087). The +CO2+O3 treatment led to greater elevation than that observed in control and the +CO2 treatment group. In allometric assessments, the regression slopes in relationships between whole plant and leaf weights (p = 0.013) and between whole plant and shoot weights (p = 0.032) differed significantly between treatments. The slopes (p = 0.635) and magnitudes (p = 0.725) of the allometric relationships between whole plant and stem weights did not differ.

4. Discussion

The present screen-aided FACE experiment demonstrated that the growth of F. crenata was enhanced by O3 exposure under elevated CO2, but not under ambient CO2 (Table 2). The increased biomass of F. crenata seedling under +CO2+O3 treatment might reflect (1) an alleviation of the negative effect of O3 on photosynthetic activity (leaf level response; Figure 1, Table 1), and (2) an increase in biomass allocation into leaves at the expense of root growth (plant-level responses; Figure 3, Table 4). The incremental increase in the biomass of F. crenata under +CO2+O3 was less than those reported in two Quercus species grown under elevated CO2 and O3 [45]. Watanabe et al. [50] also reported incremental increases in plant biomass of F. crenata in open top chamber (OTC) experiments with elevated CO2 and O3 concentrations. Our field experiment confirmed that elevated CO2 ameliorated the negative effects of elevated O3 in F. crenata trees, which are highly susceptible to increased O3 levels.
The positive effects by elevated CO2 on the whole plant biomasses of F. crenata seedlings were shown in this relatively short-term experiment (Table 2). It is obvious that the responses to elevated CO2 are dependent on the duration of exposure. Agathokleous et al., [62] reported that 11 years of exposure of CO2 in the FACE system, established in the nursery of Hokkaido University (Sapporo, Japan), induced rhizomorphogenesis, with a massive production of fine roots, especially in the phosphorus-limited volcanic ash soils, but not in the brown forest soils. The observations from different systems and experimental setups suggest that the effect of elevated CO2 on F. crenata would be dependent on soil conditions.
Fagus crenata seedlings grown under +CO2+O3 showed the lowest gsw-growth-CO2 values among the all treatment combinations in the later growing season (Figure 1B), suggesting a reduction in O3 uptake under the conditions of +CO2+O3. Hence, as a leaf level photosynthetic response, the negative effects of O3 on Agrowth-CO2 of F. crenata might be alleviated by higher CO2 levels due to reduced O3 uptake (Figure 1A). Elevated CO2–induced decreases in stomatal conductance were also reported in F. crenata and other species under high CO2 and O3 levels [50,51]. Compensation for O3-induced reductions in area-based CO2 uptake rates may partly reflect lowered O3 uptake rates due to stomatal narrowing in response to elevated CO2 [21,52]. Several studies on broadleaf tree species have reported similar effects of elevated CO2 alleviating O3-related foliar injury [21,51].
Plants grown under +CO2+O3 showed the highest LMA throughout growth season (Figure 2B). Plants with higher LMA reportedly have high tolerance to elevated O3 [3,15,27], which may also decrease concentrations of total nonstructural carbohydrates (TNC; soluble sugar and starch) and alter their distributions among plant tissues [24]. On contrast, TNC is usually increased in leaves under elevated CO2 conditions, which enhance LMA [63]. Ozone damages leaf cells and thus adversely affects plant production, reduces photosynthetic rates, and demands increased resource allocation to detoxify and repair leaves [6]. Lindroth [8] suggested that enriched in CO2 concentrations can increase carbon availability for the production of defense compounds pathways that are upregulated in response to O3. Matsumura et al. [51] and Karonen et al. [64] showed that condensed tannins are present at significantly higher levels in seedlings of F. crenata after combined treatments with high CO2 and O3 levels. The changes in TNC and these defense compounds may contribute to increased LMA in F. crenata under combined increases in CO2 and O3 levels, yet we did not determine both of them in the present study.
Elevated O3 can impair C metabolism and decrease C stocks [24], and several studies indicate decreases in biomass allocation into roots [26]. In the plant-level responses of F. crenata seedlings shown herein, allometric relationships between whole plant weight and root weight, and between roots and leaves were modified by +CO2+O3 treatments (Figure 3, Table 4). This suggests that F. crenata seedlings grown under the +CO2+O3 allocated larger amounts of biomass to aboveground tissues than belowground tissues, resulting in higher S:R ratio and L:R ratio, and in lower RWR compared with other treatments (Table 3). Reduced C allocation into root has also been demonstrated in young [16,30,65] and mature trees of Fagus species under conditions of elevated O3 [19]. However, in mature Fagus species, different responses to elevated O3 were also observed, such as stimulation of root growth [66,67] and no changes in carbon allocation into belowground tissues [68]. In addition, soil nutrient supply mitigated the negative impacts of O3 on biomass allocation in F. crenata seedlings [69,70].
In our study, all F. crenata seedlings grown under +CO2+O3 produced second-flush shoots. In addition, +CO2+O3 led to the greatest average length of the secondary shoots among the treatment combinations, although F. crenata is a determinant tree species that usually flushes shoots once a year. Watanabe et al. [50] also showed similar growth responses with enhanced second flushes of F. crenata under elevated CO2 and O3 in OTC experiments. This suggests that the greater investment of carbohydrate due to the higher photosynthetic rate in first flush leaves contributed to the increase in new leaf emergence. Kolb and Matyssek [71] similarly showed that plants with more determinate growth habits tend to respond to O3 exposure by reducing carbon allocation to root growth, in favor of maintaining older leaves and supporting new leaf flushes. Sitch et al. [20] also suggested that translocation patterns to different plant organs might depend as much on sink activities as well as source strengths. In our study, the balance between higher source activity (Agrowth-CO2) due to increased CO2 concentration, and higher sink strength for detoxification and repair of leaves following O3 exposure, might be responsible for the changes in allometric relationships between roots and leaves. Conversely, lower Agrowth-CO2 and smaller numbers of second-flush leaves in F. crenata seedlings of the +O3 treatment group may underlie the comparatively unclear changes in biomass allocation.
We observed declines in Fv/Fm ratios of F. crenata under elevated O3 later in the growth season (Table 1), suggesting that autumn leaf senescence was accelerated by O3 exposure. Watanabe et al. [72] performed FACE experiments and showed ozone-induced reductions in light-saturated photosynthetic rates in upper canopy leaves of F. crenata during August and October, but not during July. Grams et al. [21] also reported marked declines in photosynthetic rate responses to O3 in Fagus sylvatica during the late season. In addition, even current ambient atmospheric O3 levels have accelerated autumn senescence in a forest of F. crenata in the northern part of Japan [49]. These dynamic interactions between plant development, carbon allocation, and O3 exposure are important for understanding future impacts of ozone on forest ecosystems [6]. The results of our screen-aided free-air O3 and CO2 exposure experiments in the field may contribute to predictions of tree responses to future climate change.
Since both CO2 and O3 are categorized global-warming-gases, along with increased O3 and CO2 concentrations, ambient air temperature may increase in the future. However, our study did not treat air temperature, and the air temperature in each frame was not affected by CO2 and O3 treatments. Hence, this study focused on the effects of increased CO2 and O3 without consideration of temperature.

5. Conclusions

Screen-aided FACE experiments demonstrated that the growth of Fagus crenata seedlings was enhanced by combined increases in O3 and CO2 concentrations, which was related to the modified C allocation between roots and leaves (plant-level responses), and the alleviation of O3 impact on net CO2 assimilation by elevated CO2 (leaf level responses). It is noteworthy that the extent of growth enhancement was not as large as that reported in Quercus species [45]. The intrinsic lower plasticity of leaf expansion pattern in F. crenata would affect the growth responses to future combinations of increased O3 and CO2 levels.

Author Contributions

H.T. and M.K. designed the study. H.T., M.K., H.H., K.Y., S.K., and M.K. collected the photosynthetic data, performed the analysis, and hence equally contributed to this study. All authors also discussed the results and commented on the manuscript.

Funding

This study was supported in part by the project on “Technology development for circulatory food production systems responsive to climate change” conducted by the Ministry of Agriculture, Forestry and Fisheries, Japan, the Grant-in-Aid for Scientific Research (B) (No. 25292092) and JSPS KAKENHI Grant Number JP17H03839.

Acknowledgments

We thank K Sakai and K. Arai for assistance with measurements. We also thank Morikawa for his valuable suggestions concerning the present study.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Seasonal changes in the light-saturated net CO2 assimilation rate (Agrowth-CO2) (A), stomatal conductance (gsw-growth-CO2) (B), and intercellular CO2 concentration (Ci) (C) in the first flush leaves of F. crenata seedlings grown under the CO2 and O3 treatment combinations at each ambient CO2 level (380 and 550 µmol mol−1). Diamond: control, square: elevated CO2, triangle: elevated O3, circle: the combination of elevated CO2 and O3. Dotted line shows the period of the bud break. The effects (p < 0.05) of CO2 treatments, O3 treatments, and the interaction CO2 and O3 on each parameter for each measurement period (five times) are indicated in the panel by CO2, O3, and CO2 × O3, respectively. The parenthesis indicate difference at p < 0.1, and n.s. indicate nonsignificant (p > 0.1). Values are means ± SE (n = 3).
Figure 1. Seasonal changes in the light-saturated net CO2 assimilation rate (Agrowth-CO2) (A), stomatal conductance (gsw-growth-CO2) (B), and intercellular CO2 concentration (Ci) (C) in the first flush leaves of F. crenata seedlings grown under the CO2 and O3 treatment combinations at each ambient CO2 level (380 and 550 µmol mol−1). Diamond: control, square: elevated CO2, triangle: elevated O3, circle: the combination of elevated CO2 and O3. Dotted line shows the period of the bud break. The effects (p < 0.05) of CO2 treatments, O3 treatments, and the interaction CO2 and O3 on each parameter for each measurement period (five times) are indicated in the panel by CO2, O3, and CO2 × O3, respectively. The parenthesis indicate difference at p < 0.1, and n.s. indicate nonsignificant (p > 0.1). Values are means ± SE (n = 3).
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Figure 2. Seasonal change in the leaf N concentration (leaf Nmass) (A), leaf mass per area (LMA) (B), and area-based leaf N content (leaf Nmass) (C) in the first flush leaves of F. crenata seedlings grown under the CO2 and O3 treatment combinations at each ambient CO2 level (380 and 550 µmol mol−1). The format of these figures is the same as in Figure 1.
Figure 2. Seasonal change in the leaf N concentration (leaf Nmass) (A), leaf mass per area (LMA) (B), and area-based leaf N content (leaf Nmass) (C) in the first flush leaves of F. crenata seedlings grown under the CO2 and O3 treatment combinations at each ambient CO2 level (380 and 550 µmol mol−1). The format of these figures is the same as in Figure 1.
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Figure 3. Allometric relationships between whole plant weight and root weight (A) and between root weight and leaf weight (B) of F. crenata seedlings. Allometric equations analyzed by using an R package, smart 3 (SMA analysis), are listed in Table 4. Diamond (black): control, square (blue): elevated CO2, triangle (green): elevated O3, circle (red): the combination of elevated CO2 and O3. Each colored line shows regression line for each treatment.
Figure 3. Allometric relationships between whole plant weight and root weight (A) and between root weight and leaf weight (B) of F. crenata seedlings. Allometric equations analyzed by using an R package, smart 3 (SMA analysis), are listed in Table 4. Diamond (black): control, square (blue): elevated CO2, triangle (green): elevated O3, circle (red): the combination of elevated CO2 and O3. Each colored line shows regression line for each treatment.
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Table
Table 1. Light-saturated net CO2 assimilation rate (Agrowth-CO2) and stomatal conductance (gsw-growth-CO2) in May and July at each ambient CO2 level (380 and 550 µmol mol−1), the maximum rate of carboxylation at 25 °C (Vcmax25) in August, and the maximum PS II photochemical efficiency (Fv/Fm) at predawn in October in the first flush leaves of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. Significant effects are indicated by *; p < 0.05, and n.s.; non-significant.
Table 1. Light-saturated net CO2 assimilation rate (Agrowth-CO2) and stomatal conductance (gsw-growth-CO2) in May and July at each ambient CO2 level (380 and 550 µmol mol−1), the maximum rate of carboxylation at 25 °C (Vcmax25) in August, and the maximum PS II photochemical efficiency (Fv/Fm) at predawn in October in the first flush leaves of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. Significant effects are indicated by *; p < 0.05, and n.s.; non-significant.
TreatmentsAgrowth-CO2 (µmol m−2 s−1)gsw-growth-CO2 (µmol m−2 s−1)Vcmax25 (µmol m−2 s−1)Predawn-Fv/Fm
(May)(July)(May)(July)(August)(October)
Control8.4 ± 0.810.8 ± 1.60.12 ± 0.010.23 ± 0.0230.1 ± 3.50.76 ± 0.01
+CO29.4 ± 0.312.7 ± 1.30.09 ± 0.010.18 ± 0.0223.0 ± 2.10.78 ± 0.01
+O38.6 ± 0.6 9.3 ± 1.40.12 ± 0.010.27 ± 0.0121.9 ± 2.00.65 ± 0.04
+CO2+O310.1 ± 0.712.9 ± 1.10.09 ± 0.010.22 ± 0.0222.2 ± 2.80.72± 0.01
Effect
CO2 (F1,8)3.7 n.s.4.0 n.s.6.34 *8.94 *1.65 n.s.0.08 n.s.
O3 (F1,8)0.5 n.s.0.2 n.s.0.05 n.s.4.90 n.s.2.86 n.s.2.97 n.s.
CO2 × O3 (F1,8)0.2 n.s.0.3 n.s.0.00 n.s.0.01 n.s.1.93 n.s.0.03 n.s.
Table
Table 2. Dry mass of plant tissues (whole plant; Total, leaf weight; Leaf, current shoot weight; Current shoot; stem and shoot weight; Stem and shoot, root weight; Root) of the seedling of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. Significant effects are indicated by *; p < 0.05, and n.s.; non-significant.
Table 2. Dry mass of plant tissues (whole plant; Total, leaf weight; Leaf, current shoot weight; Current shoot; stem and shoot weight; Stem and shoot, root weight; Root) of the seedling of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. Significant effects are indicated by *; p < 0.05, and n.s.; non-significant.
TreatmentsTotalLeafCurrent ShootStem and ShootRoot
Control43.3 ± 9.65.5 ± 1.58.8 ± 3.28.7 ± 1.320.3 ± 3.7
+CO245.4 ± 6.45.3 ± 1.310.0 ± 2.610.2 ± 1.619.9 ± 2.2
+O339.2 ± 0.75.4 ± 0.56.9 ± 0.38.9 ± 1.218.0 ± 0.9
+CO2+O367.1 ± 1.39.2 ± 0.416.4 ± 0.615.1 ± 1.326.4 ± 0.8
Effect
CO2 (F1,8)6.6 *3.1 n.s.6.5 *7.8 *3.2 n.s.
O3 (F1,8)2.3 n.s.3.4 n.s.1.2 n.s.3.5 n.s.0.9 n.s.
CO2 × O3 (F1,8)4.9 n.s.3.8 n.s.3.9 n.s.2.9 n.s.3.8 n.s.
Table
Table 3. Dry matter distribution (leaf weight ratio; LWR, current shoot weight ratio; CSWR, stem and shoot weight ratio; SWR, root weight ratio; RWR, and aboveground biomass to root ratio; S:R ratio, leaf to root ratio; L:R ratio) of the seedling of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. There is no significant effect in all parameters, which indicated by n.s.; non-significant.
Table 3. Dry matter distribution (leaf weight ratio; LWR, current shoot weight ratio; CSWR, stem and shoot weight ratio; SWR, root weight ratio; RWR, and aboveground biomass to root ratio; S:R ratio, leaf to root ratio; L:R ratio) of the seedling of F. crenata grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values at upper half lines are means ± SE of three replicates for each treatment. F values of analysis of variance (ANOVA) to test the main effects of CO2, O3, and their interactions are also shown at lower half lines. There is no significant effect in all parameters, which indicated by n.s.; non-significant.
TreatmentsLWRCSWRSWRRWRS:R RatioL:R Ratio
Control0.12 ± 0.010.19 ± 0.030.21 ± 0.020.48 ± 0.021.12 ± 0.090.26 ± 0.04
+CO20.12 ± 0.020.21 ± 0.030.23 ± 0.030.44 ± 0.021.30 ± 0.110.26 ± 0.04
+O30.14 ± 0.010.18 ± 0.000.23 ± 0.030.45 ± 0.021.25 ± 0.130.30 ± 0.02
+CO2+O30.14 ± 0.010.24 ± 0.010.21 ± 0.010.41 ± 0.011.50 ± 0.060.35 ± 0.02
Effect
CO2 (F1,8)0.01 n.s.2.84 n.s.0.03 n.s.4.07 n.s.4.52 n.s.0.82 n.s.
O3 (F1,8)3.37 n.s.0.09 n.s.0.01 n.s.2.24 n.s.2.65 n.s.4.57 n.s.
CO2 × O3 (F1,8)0.05 n.s.0.82 n.s. 0.65 n.s.0.08 n.s.0.18 n.s.0.91 n.s.
Table
Table 4. Standardized major axis (SMA) regression slopes and their confidence intervals for log-log transformed relationships between whole plant weight and root weight (Figure 3A) and between root weight and leaf weight (Figure 3B) of F. crenata seedlings grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values of coefficients of determination (r2) and significant values (p) of each bivariate relationship are shown. 95% confidence intervals (CI) of SMA slopes and y-axis intercepts are exhibited. In cases where SMA tests for common slopes revealed no significant differences between treatments, common slopes are used. The differences in elevations of the SMA regressions are tested among treatments using Sidak’s adjusted pair-wise test, depending on the significance of the null hypothesis that the slopes are equal. Where there is a significant difference in elevation of the common-slope SMA regressions, values for the y-axis intercept (elevation) are provided. Different letters in pairwise comparison are significantly different, with a p < 0.05 under the slopes are equal. Where appropriate, significant shifts along a common slope are indicated. The format of table is referred that in Atkin et al. [59].
Table 4. Standardized major axis (SMA) regression slopes and their confidence intervals for log-log transformed relationships between whole plant weight and root weight (Figure 3A) and between root weight and leaf weight (Figure 3B) of F. crenata seedlings grown under ambient air (control), elevated CO2 (+CO2), elevated O3 (+O3), and the combination of elevated CO2 and O3 (+CO2+O3). Values of coefficients of determination (r2) and significant values (p) of each bivariate relationship are shown. 95% confidence intervals (CI) of SMA slopes and y-axis intercepts are exhibited. In cases where SMA tests for common slopes revealed no significant differences between treatments, common slopes are used. The differences in elevations of the SMA regressions are tested among treatments using Sidak’s adjusted pair-wise test, depending on the significance of the null hypothesis that the slopes are equal. Where there is a significant difference in elevation of the common-slope SMA regressions, values for the y-axis intercept (elevation) are provided. Different letters in pairwise comparison are significantly different, with a p < 0.05 under the slopes are equal. Where appropriate, significant shifts along a common slope are indicated. The format of table is referred that in Atkin et al. [59].
ResponseWhole Plant WeightLeaf Weight
BivariateRoot WeightRoot Weight
 H0 NO. 1: no difference in slope (p-value)
0.2310.114
TreatmentControl+CO2+O3+CO2+O3Control+CO2+O3+CO2+O3
n1515222115152221
r20.8590.9170.9460.9520.3930.3670.6260.751
p-value<0.0001<0.0001<0.0001<0.00010.0120.017<0.0001<0.0001
Slope0.9611.0181.1070.9521.5791.1520.8910.887
Slope CI_high1.2021.2081.2331.0582.4801.8261.1801.125
Slope CI_Low0.7680.8580.9930.8571.0060.7270.6720.700
Intercept−0.257−0.392−0.509−0.315−0.592−0.766−0.385−0.304
 H0 NO. 2: no difference in elevation (p-value)
0.00020.009
Intercepts for a common slope−0.343−0.387−0.368−0.427−0.592−0.544−0.492−0.431
 Pairwise comparison (where relationship significant)
aababbbaba
 H0 NO. 3: no difference in ’shift’ (p-value)
0.0150.002

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Tobita, H.; Komatsu, M.; Harayama, H.; Yazaki, K.; Kitaoka, S.; Kitao, M. Effects of Combined CO2 and O3 Exposures on Net CO2 Assimilation and Biomass Allocation in Seedlings of the Late-Successional Fagus Crenata. Climate 2019, 7, 117. https://0-doi-org.brum.beds.ac.uk/10.3390/cli7100117

AMA Style

Tobita H, Komatsu M, Harayama H, Yazaki K, Kitaoka S, Kitao M. Effects of Combined CO2 and O3 Exposures on Net CO2 Assimilation and Biomass Allocation in Seedlings of the Late-Successional Fagus Crenata. Climate. 2019; 7(10):117. https://0-doi-org.brum.beds.ac.uk/10.3390/cli7100117

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Tobita, Hiroyuki, Masabumi Komatsu, Hisanori Harayama, Kenichi Yazaki, Satoshi Kitaoka, and Mitsutoshi Kitao. 2019. "Effects of Combined CO2 and O3 Exposures on Net CO2 Assimilation and Biomass Allocation in Seedlings of the Late-Successional Fagus Crenata" Climate 7, no. 10: 117. https://0-doi-org.brum.beds.ac.uk/10.3390/cli7100117

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