Growth and Photosynthetic Responses of Seedlings of Japanese White Birch, a Fast-Growing Pioneer Species, to Free-Air Elevated O₃ and CO₂

Abstract

Plant growth is not solely determined by the net photosynthetic rate (A), but also influenced by the amount of leaves as a photosynthetic apparatus. To evaluate growth responses to CO₂ and O₃, we investigated the effects of elevated CO₂ (550–560 µmol mol⁻¹) and O₃ (52 nmol mol⁻¹; 1.7 × ambient O₃) on photosynthesis and biomass allocation in seedlings of Japanese white birch (Betula platyphylla var. japonica) grown in a free-air CO₂ and O₃ exposure system without any limitation of root growth. Total biomass was enhanced by elevated CO₂ but decreased by elevated O₃. The ratio of root to shoot (R:S ratio) showed no difference among the treatment combinations, suggesting that neither elevated CO₂ nor elevated O₃ affected biomass allocation in the leaf. Accordingly, photosynthetic responses to CO₂ and O₃ might be more important for the growth response of Japanese white birch. Based on A measured under respective growth CO₂ conditions, light-saturated A at a light intensity of 1500 µmol m⁻² s⁻¹ (A₁₅₀₀) in young leaves (ca. 30 days old) exhibited no enhancement by elevated CO₂ in August, suggesting photosynthetic acclimation to elevated CO₂. However, lower A₁₅₀₀ was observed in old leaves (ca. 60 days old) of plants grown under elevated O₃ (regulated to be twice ambient O₃). Conversely, light-limited A measured under a light intensity of 200 µmol m⁻² s⁻¹ (A₂₀₀) was significantly enhanced by elevated CO₂ in young leaves, but suppressed by elevated O₃ in old leaves. Decreases in total biomass under elevated O₃ might be attributed to accelerated leaf senescence by O_3, indicated by the reduced A₁₅₀₀ and A₂₀₀ in old leaves. Increases in total biomass under elevated CO₂ might be attributed to enhanced A under high light intensities, which possibly occurred before the photosynthetic acclimation observed in August, and/or enhanced A under limiting light intensities.

1. Introduction

Atmospheric CO₂ concentration is increasing globally [1,2], accompanied by an increase in tropospheric ozone (O₃) concentration, particularly in East Asia [3,4,5,6]. O₃ pollution reduces plant growth and productivity through a reduction in photosynthesis, an increase in leaf respiration and acceleration of leaf senescence [7,8,9,10,11]. O₃ exposure reduces photosynthetic rate by a reduction in the maximum rate of Rubisco carboxylation (V_c,max) [12,13,14,15] as well as a decrease in stomatal conductance [14,16,17]. Conversely, elevated CO₂ generally increases plant growth with an enhancement of photosynthesis [18,19,20]. However, long-term elevated CO₂ leads to photosynthetic acclimation accompanied by a decrease in the Rubisco carboxylation capacity, indicated by a decrease in V_c,max (so-called “photosynthetic downregulation”) frequently accompanied by leaf starch accumulation and leaf nitrogen reduction [19,21].

Plant growth is not solely determined by photosynthetic rate, usually expressed as the rate of leaf-area-based CO₂ assimilation, but is also influenced by the amount of leaf area as a photosynthetic apparatus [22]. An increase in the shoot to root ratio (S:R ratio) was observed in some plants grown under elevated O₃ [23,24,25], which is considered a compensatory response against O₃-induced photosynthesis reduction by investing biomass into the photosynthetic apparatus [26].

Deciduous broadleaf forests are broadly distributed in Japan, including various tree species with different morphological and physiological traits along with forest succession [27,28]. Early successional species, such as birch species, have a succeeding-type shoot development, whereas mid- and late-successional species, such as Japanese oak (Quercus mongolica var. crispula) and Siebold’s beech (Fagus crenata), have a flush-type shoot development, generally flushing once a year in spring. Consequently, early successional pioneer species have a relatively higher S:R ratio than mid- and late-successional species (Shukla and Ramakrishnan, 1984; Kitao et al., 2005, 2015). Such species-specific differences in the shoot development pattern might influence the growth responses to elevated O₃ and CO₂.

A significant growth enhancement was previously observed in seedlings of two mid-successional species (Q. mongolica var. crispula and Q. serrata) and late successional species (F. crenata) grown under the combination of elevated O₃ and CO₂ [29,30]. These tree species exhibited an increased S:R ratio under O₃ exposure, with stimulation of new shoot development and enhanced photosynthetic rate under elevated CO₂.

As an early successional species, Japanese white birch has a preferable biomass allocation into shoots with successional leaf development [27,31]. We hypothesized that the intrinsically higher S:R ratio in Japanese white birch would restrict the compensatory response against O₃ exposure via a shift of biomass allocation into shoot. In this context, photosynthetic responses to O₃ and CO₂ exposure might directly affect the growth response of the pioneer Japanese white birch. To test this hypothesis, we investigated photosynthetic properties, growth, and biomass allocation in seedlings of Japanese white birch grown in a free-air, CO₂ and O₃ exposure system.

2. Materials and Methods

2.1. Plant Materials

Bare-rooted, 1 year old seedlings of Japanese birch (Betula platyphylla var. japonica) obtained from a commercial nursery (Hokkaido Engei-Ryokka Center, Kitahiroshima, Hokkaido) were planted directly in soil inside the frames of a free-air CO₂ and O₃ exposure system that was established in the nursery of Forestry and Forest Products Research Institute in Tsukuba, Japan (36°00′ N, 140°08′ E, 20 m a.s.l.) in March 2011. Twelve frames (2 × 2 × 2 m), partly surrounded by transparent windscreens (15 to 65 cm, and 75 to 125 cm in height above the soil) to reduce wind while not interfering with air exchange inside–outside, were installed for free-air CO₂ and O₃ enrichment. The free-air, CO₂ and O₃ exposure system is described in detail in [29]. The height of the seedlings was ca. 15 cm. Six seedlings were planted within each frame (totally 72 plants in the 12 frames). The CO₂ and O₃ treatments were as follows: control (unchanged ambient air), elevated CO₂ (eCO₂, target: 550 µmol mol⁻¹), elevated O₃ (eO₃, target: 2.0 × ambient O₃), and elevated CO₂ + O₃ (eCO₂ + eO₃, 550 µmol mol⁻¹ CO₂ and 2.0 × ambient O₃). The twelve frames were divided into three replicates per gas treatment. The treatments lasted from the beginning of May to the middle of November 2011 (

Table 1. CO₂ and O₃ concentrations in the free-air fumigation system. Values are means ± SD (n = 3) of daytime CO₂ and O₃ concentrations (6:00 to 18:00) during the growth season (May to November 2011).

Treatment	Control	eCO2	eO3	eCO2 + eO3
CO2 (µmol mol−1)	377 ± 1.0	563 ± 9.0	381 ± 1.2	546 ± 2.3
O3 (nmol mol−1)	30.2 ± 0.4	30.2 ± 0.7	52.2 ± 2.1	51.6 ± 3.1

). During the experimental period, we had periodic precipitation (Figure 1), thus we only irrigated in the case of prolonged sunny days with no precipitation.

2.2. Measurements of Gas Exchange

Gas exchange measurements were conducted in the beginning of August 2011, for randomly selected young (≈30 day old) and old (≈60 day old) leaves of a representative seedling per frame, i.e., three replications for each treatment combination (CO₂ × O₃), using a portable photosynthesis system (Model LI-6400; LI-COR, Lincoln, NV, USA). A total of 12 seedlings were selected, and one young leaf (≈13th leaf position) and one old leaf (≈7th leaf position from the bottom leaf of main shoot) in the same seedling were used (a total of 24 leaves). Japanese birch has heterophyllous leaves (i.e., early leaves flushed in spring and late leaves succeedingly developed during summer) [15,31]. We used late leaves of different ages (young and old), where young leaves were flushed in July and old leaves flushed in June. Because of the succeeding-type leaf development, both young and old leaves were developed under a sunlit condition—considered sun leaves.

Measurements were conducted between 8:00 and 16:00. As the sunrise was around 4:30, and the sunset was around 19:00 in the beginning of August, we considered that the leaves had been photosynthetically activated under sunlight at the onset of the gas exchange measurements. The CO₂ response evaluation was conducted as follows: at a saturating photon flux density (PFD) of 1500 µmol m⁻² s⁻¹, we first measured the net photosynthetic rate (A) at a CO₂ concentration of 100 µmol mol⁻¹ after 15 min acclimation, then measured A at CO₂ concentrations of 200, 380, 550, and 1000 µmol mol⁻¹ in sequence after ≈5 min acclimation for each CO₂ concentration. Measurements were conducted under a block temperature of 27 °C (leaf temperature, 31.5 ± 0.3 °C, mean ± SE, n = 24) and a relative humidity of ≈80%. We monitored A, stomatal conductance (g_s), and intercellular CO₂ concentration (C_i) throughout the measurements and recorded their values after they reached the steady state. We then investigated light-acclimated photosynthesis as follows: after the measurement of A at a CO₂ concentration of 1000 µmol mol⁻¹, CO₂ concentration was set to the respective growth CO₂ concentration. At the respective growth CO₂ concentration, A was measured at a PFD of 2000 µmol m⁻² s⁻¹ after a 10 min acclimation. Then, A was measured at the PFDs of 1500, 1000, 600, 300, 200, and 100 µmol m⁻² s⁻¹ in sequence after a 4 min acclimation for each PFD. We considered A measured at a PFD of 1500 under the respective growth CO₂ concentrations (i.e., CO₂ concentration of 380 µmol mol⁻¹ for control and eO₃ treatments, and 550 µmol mol⁻¹ for eCO₂ and eCO₂ + eO₃ treatments) as a measure of the light-saturated, net photosynthetic rate (A₁₅₀₀). As the quadratic equation for the light-response curve [32] failed to fit in some cases, especially for old leaves, we used A measured at a PFD of 200 µmol m⁻² s⁻¹ under the respective growth CO₂ concentration as a direct measure of the light-limited photosynthetic rate (A₂₀₀).

We used a traditional, steady-state A/C_i response technique [33] but with fewer CO₂ points. The maximum rates of Rubisco carboxylase (V_c,max) and RuBP regeneration (J_max) were estimated based on the protocol using linear regression in [33]. In the case that A is limited by Rubisco carboxylation, A is expressed as A = V_c,max f’ − R_d, where f’ = $\frac{C_i-{\mathsf{\Gamma }}^{*}}{C_i+K_c\left(1+\frac{O}{K_o}\right)}$, and R_d is mitochondrial respiration under light. Г* is the CO₂ compensation point, K_c Rubisco Michaelis constant for CO₂, K_o Rubisco Michaelis constant for O₂, and O oxygen concentration in air. Г*, K_c, and K_o at the leaf temperature during gas exchange measurements were estimated based on the temperature responses of them [34]. V_c,max is estimated as the slope of the A and f’ relationship, and R_d is estimated as the y-intercept. We used A at the CO₂ concentrations of 100, 200, and 380 µmol mol⁻¹ for V_c,max and R_d estimation, as A is generally limited by Rubisco carboxylation under ambient CO₂ and saturated light [35,36]. Furthermore, we confirmed the linearity of the three points of A as a function of f’ (Supplemental Figure S1), supporting that A at 380 µmol mol⁻¹ CO₂ was limited by Rubisco carboxylation.

Similarly, when A is limited by RuBP regeneration under saturating light, J_max is determined as the slope of the following equation: A = J_max g’ − R_d, where g’ = $\frac{C_i-{\mathsf{\Gamma }}^{*}}{4.5 C_i+10.5 {\mathsf{\Gamma }}^{*}}$. In this case, R_d was determined as explained above [33]. A measured at the CO₂ concentrations of 550 and 1000 µmol mol⁻¹ was used for the J_max determination since linearity was observed in the relationship between A+R_d at these CO₂ concentrations and g’ through the origin (A + R_d = J_max g’) (Supplemental Figure S1). This supports that A at 550 and 1000 µmol mol⁻¹ CO₂ was limited by RuBP regeneration.

Notably, g_s stayed above 0.08 mol m⁻² s⁻¹ during the A/C_i measurements, which is the threshold of accurate A/C_i response measurement, regarding stomatal patchiness [37]. V_c,max and J_max, estimated at the leaf temperature of ≈32 °C, were normalized to those at 25 °C by using their temperature responses according to [34].

2.3. Growth and Biomass Allocation

At the end of the experiments (November 2011), all seedlings were harvested. The soils were carefully removed by hand, using small, stainless-steel rakes (Bonsai rake, Kikuwa, Sanjo, Niigata, Japan). The biomasses of the leaves, stems, and roots were measured after oven-drying at 70 °C to a constant weight. We also collected all leaves that were shed before harvest. Senescent leaves with an abscission layer, which were easily detached by hand, were collected every day from 1 September 2011 to the end of the experiments; we considered them shed leaves. We calculated the ratio of shoot to root (S:R ratio), defined as [leaf + shed leaf + stem dry mass]/[root dry mass], and leaf weight ratio (LWR), calculated as [leaf + shed leaf dry mass]/[total dry mass]. As some seedlings died during the growth season irrespective of the treatment, due to some unidentified reason, 7 to 14 plants per treatment combination were sampled, where initially 18 plants were planted per treatment combination (totally 72 plants).

2.4. Leaf Nitrogen Content

The area-based leaf nitrogen content (N_area) was determined for the leaves used for the gas exchange measurements 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). The leaf area of the sampled whole leaves was determined, using a scanner (LiDE210, Canon, Tokyo, Japan) and an image analysis software (LIA32 ver 0.3781, http://www.agr.nagoya-u.ac.jp/~shinkan/LIA32/, accessed on 24 May 2021). The leaf mass per area (LMA = leaf dry mass/leaf area) was calculated from the leaf area and leaf dry mass measured after oven-drying at 70 °C. Using LMA, dry-mass-based leaf N was converted to N_area.

2.5. Statistical Analysis

As one representative plant per FACE frame (experimental unit; n = 3 for each treatment combination) was used for the gas exchange measurements and leaf N analysis, a two-factorial ANOVA was used to test the effects of CO₂, O₃ and their interaction on the photosynthetic properties and leaf N content of young and old leaves (R Development Core Team 2014). A linear mixed model was applied to analyze the total biomass and S:R ratio, with CO₂ and O₃ treatments as fixed factors and the frame as a random effect. We used the lmer function of the R package lme4 for the model fitting [38], and the ANOVA function of the R package car for the analysis of the deviance table [39]. The level of significance was 0.05.

3. Results

3.1. Growth Responses of Japanese white Birch Seedlings to Elevated CO₂ and O₃

The total biomass was increased by the elevated CO₂ but decreased by the elevated O₃ (Figure 2a). Conversely, the S:R ratio showed no significant difference among the treatment combinations (Figure 2b). Similar to the S:R ratio, LWR showed no significant difference among the treatment combinations (Figure 2c). The shed leaf to total leaf dry mass, defined as [shed leaf dry mass]/[leaf + shed leaf dry mass], was significantly increased by the elevated O₃ (Figure 2d).

3.2. Net Photosynthetic Rate and Stomatal Conductance in Young and Old Leaves of Japanese Birch Seedlings Grown under the Combination of CO₂ and O₃

The light-saturated net photosynthetic rate under a light intensity of 1500 µmol m⁻² s⁻¹ (A₁₅₀₀) showed no significant difference in young leaves (≈ 30 day old) among the treatment combinations, whereas decreases in A₁₅₀₀ were observed in old leaves (≈ 60 day old) of plants grown under elevated O₃ (Figure 3, upper panel). Conversely, the light-limited net photosynthetic rate, measured under a light intensity of 200 µmol m⁻² s⁻¹ (A₂₀₀), showed higher values in young leaves of plants grown under elevated CO₂, whereas elevated O₃ decreased A₂₀₀ in old leaves (Figure 3, lower panel). In all cases, no significant effect of the CO₂ × O₃ interaction was observed. Stomatal conductance under a saturating light intensity of 1500 µmol m⁻² s⁻¹ (g_s1500) showed no significant difference among the treatment combinations both in young and old leaves (Figure 4, upper panel). Although stomatal conductance under a limiting light intensity of 200 µmol m⁻² s⁻¹ (g_s200) showed no significant difference in young leaves, g_s200 was significantly decreased by elevated O₃ (Figure 4, lower panel).

3.3. Maximum Rates of Rubisco Carboxylation (V_c,max) and RuBP Regeneration (J_max) in Young and Old Leaves of Japanese Birch Seedlings Grown under the Combination of CO₂ and O₃

The maximum rate of Rubisco carboxylation (V_c,max) was decreased by the elevated CO₂ in young leaves, whereas V_c,max was decreased by both the elevated CO₂ and O₃ in older leaves (Figure 5, upper panel). The maximum rate of RuBP regeneration (J_max) was also decreased by the elevated CO₂ in young leaves and decreased by the elevated CO₂ and O₃ in old leaves (Figure 5, lower panel). No significant effect of the CO₂ × O₃ interaction on V_c,max and J_max was observed.

3.4. Leaf Nitrogen Content

The area-based leaf nitrogen content (N_area) generally decreased in older leaves, while leaves of plants grown under elevated CO₂ showed significantly lower N_area for both young and old leaves of Japanese white birch seedlings (Figure 6). Conversely, no significant effect of the CO₂ × O₃ interaction was observed.

4. Discussion

In the present study, we observed growth enhancement by elevated CO₂ but suppression by elevated O₃ in Japanese white birch seedlings grown in a free-air CO₂ and O₃ exposure system without any limitation of root growth as it has been reported for the pioneer tree species Populus tremuloides [40]. However, the growth responses in Japanese white birch were quite different from those observed in mid- and late-successional tree species. Mid-successional tree species (Japanese oak (Quercus mongolica var. crispula) and Konara oak (Q. serrata)), and late-successional tree species (Sebold’s beech (Fagus crenata)), showed an enhancement of shoot growth relative to root growth by O₃ exposure, which fully compensated the reduction in the photosynthetic rate by O₃ regarding plant growth, and even led to significant increases in total dry mass under the combination of elevated CO₂ and O₃ [29,30,41]. Thus, growth responses to elevated CO₂ and O₃ might depend on both photosynthetic responses and biomass allocation into plant organs, especially into leaves.

Japanese white birch seedlings showed little change in biomass allocation as is indicated by the almost constant value of the S:R ratio (around 2) and leaf weight ratio (≈0.23) (Figure 2). As a pioneer tree species, Japanese white birch produces new leaves continuously throughout the growth season, while mid- and late-successional species generally flush several leaves at once in spring [27,42]. Such a difference in the shoot development pattern caused a different biomass allocation pattern, indicated by the S:R ratio; Japanese white birch showed a value of 2 but Japanese oak and Konara oak showed a value of 1 in control plants grown in ambient air [29]. Japanese white birch could not change the ratio under elevated O₃, whereas Japanese oak and Konara oak, grown in the same FACE system, changed the ratio from 1 to 2 in the case of elevated O₃ exposure [29], maybe because O₃-induced hormonal changes might stimulate the intrinsically conservative shoot growth in the two oak species [43,44,45]. Thus, without any changes in biomass allocation, the growth of Japanese white birch might be influenced more directly by photosynthetic responses to elevated CO₂ and O₃, in comparison with mid- or late-successional tree species.

Growth responses of Japanese white birch among the treatment combinations could not be fully explained by the differences in A₁₅₀₀ measured in mid-summer, where a higher total dry mass was observed under elevated CO₂ despite no significant enhancement in A₁₅₀₀ (Figure 2 and Figure 3). Conversely, decreases in total biomass in plants grown under O₃ exposure could be attributed to the lower A₁₅₀₀ in old leaves. Although the light-saturated photosynthetic rate is often used as a measure of plant responses to environmental stresses, diurnal changes in environmental conditions, and the leaf-age-dependent photosynthetic capacity should be taken into consideration for assessing plant growth as an integration of photosynthetic performance.

Regarding the photosynthetic capacity indicated by V_c,max and J_max, photosynthetic downregulation apparently occurred even in young leaves in August 2011, accompanied by a reduction in N_area [18,46,47]. Conversely, old leaves decreased photosynthetic capacity (V_c,max and J_max) under both elevated CO₂ and elevated O₃, suggesting that O₃-accelerated leaf senescence may be due to oxidative damage additionally occurred upon photosynthetic downregulation by the elevated CO₂ [12,13,15,48]. Elevated CO₂ can affect the photosynthetic capacity in a short term (significantly, even for young leaves), whereas O₃ only affects it over a longer term. Furthermore, although elevated O₃ did not affect leaf production (Figure 2c), O₃-accelerated leaf senescence induced earlier leaf shedding in autumn (Figure 2d), which might also cause an adverse effect on plant growth. Conversely, photosynthetic performance (indicated by A₁₅₀₀, Figure 3) of young leaves under the respective growth CO₂ concentrations showed no significant differences among the treatment combinations, suggesting that the photosynthetic downregulation (reduction in V_c,max or J_max; Figure 5) could be compensated by the elevated CO₂ by enhancing photosynthesis and suppressing photorespiration via higher intercellular CO₂ concentrations [33,48]. Such a compensative effect of the elevated CO₂ seemed still effective in old leaves, which showed lower A₁₅₀₀ only under elevated O₃.

Photosynthetic downregulation occurs with enhanced plant growth, which results from nitrogen dilution due to increased biomass over the nitrogen acquisition capacity by the root system [19,49]. In this context, the leaves of plants grown under elevated CO₂ were downregulated as of the time point of measuring photosynthesis in August 2011 as a consequence of growth enhancement, which was supported by the lower N_area in the young leaves of plants grown under elevated CO₂. Soil N availability might have been limited as of that time because of the relatively poor root system in seedlings of Japanese white birch, indicated by the high S:R ratio.

It is also noteworthy that a significantly higher light-limited photosynthetic rate (A₂₀₀) was observed in young leaves of plants grown under elevated CO₂ (Figure 3). Elevated CO₂-induced photosynthetic downregulation accompanied by decreased N_area might not influence the total electron flow under limiting light (Supplemental Figure S2). Conversely, a reduction in electron partitioning into photorespiration under elevated CO₂ resulted in a higher photosynthetic rate under limiting light (A₂₀₀) [50]. As Japanese white birch develops new leaves continuously during the growing season [27,42], most leaves are partially shaded except for the topmost ones. Accordingly, all leaves could not necessarily receive saturating light even around noon. Therefore, the higher A under limiting light in the elevated-CO₂-grown plants could also contribute to the greater growth under elevated CO₂. Such an increase in light-limited photosynthesis would be of relevance for shade tolerance among tree species with different successional traits [51].

Regarding g_s, elevated O₃ decreased g_s only in old leaves of Japanese white birch under limiting light. This suggests that O₃-induced stomatal closure, which contributes to preventing O₃ influx [17,52], might not function well in Japanese white birch. Relatively higher g_s (g_s1500, up to 0.4 mol m⁻² s⁻¹) was observed in the control plants of Japanese white birch, compared with those in Q. mongolica (up to 0.3 mol m⁻² s⁻¹) and Q. serrata (up to 0.2 mol m⁻² s⁻¹) (cf. [29]), which suggests that photosynthesis of Japanese white birch might be more sensitive to elevated O₃ exposure as a consequence of the higher phytotoxic O₃ dose through the stomata [53].

In conclusion, a pioneer species, Japanese white birch, had no compensative biomass allocation into shoot growth under elevated O₃, in contrast to mid- and late-successional species. This may be attributed to its intrinsically higher S:R ratio not allowing further changes. Accordingly, photosynthetic responses, including earlier leaf senescence, might be associated with the growth responses to elevated CO₂ and O₃. Nevertheless, age-dependent changes in photosynthetic capacity, and light-limited photosynthesis should be taken into consideration to predict growth response of Japanese white birch in future-coming atmospheric conditions.