# Effects of elevated ozone concentration and nitrogen addition on ammonia stomatal compensation point in a poplar clone

Wen Xu, Bo Shang, Yansen Xu, Xiangyang Yuan, Anthony J Dore, Yuanhong

Zhao, Raia-Silvia Massad, Zhaozhong Feng

► To cite this version:

Wen Xu, Bo Shang, Yansen Xu, Xiangyang Yuan, Anthony J Dore, et al.. Effects of elevated ozone concentration and nitrogen addition on ammonia stomatal compensation point in a poplar clone. Environmental Pollution, 2018, 238, pp.760-770. 10.1016/j.envpol.2018.03.089. hal-04353491

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1 MS Number: ENVPOL_2018_333_R1

2 **Effects of elevated ozone concentration and fertilization on ammonia stomatal compensation point in a poplar clone**

4 Wen Xu<sup>1,2</sup>, Bo Shang<sup>1,2</sup>, Yansen Xu<sup>1,2</sup>, Xiangyang Yuan<sup>1,2</sup>, Anthony J. Dore<sup>3</sup>, Yuanhong

5 Zhao<sup>4</sup>, Raia-Silvia Massad<sup>5</sup>, Zhaozhong Feng<sup>1,2*</sup>

6

7 <sup>1</sup>State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-

8 Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian

9 District, Beijing 100085, China.

10 <sup>2</sup>College of Resources and Environment, University of Chinese Academy of Sciences,

11 Beijing 100049, China.

12 <sup>3</sup>Centre for Ecology and Hydrology, Edinburgh, Bush Estate, Penicuik, Midlothian

13 EH26 0QB, UK.

14 <sup>4</sup>Laboratory for Climate and Ocean-Atmosphere Sciences, Department of Atmospheric

15 and Oceanic Sciences, School of Physics, Peking University, Beijing100871, China.

16 <sup>5</sup>UMR ECOSYS, INRA, Agroparistech, Université Paris-Saclay Thiverval-Grignon

17 France.

18 *Corresponding author.

19 E-mail address: fzz@rcees.ac.cn (Z. Feng).
**Abstract:** The stomatal compensation point of ammonia ($\chi_s$) is a key factor controlling plant-atmosphere NH$_3$ exchange, which is dependent on the nitrogen (N) supply and varies among plant species. However, knowledge gaps remain concerning the effects of elevated atmospheric N deposition and ozone (O$_3$) on $\chi_s$ for forest species, resulting in large uncertainties in the parameterizations of NH$_3$ incorporated into atmospheric chemistry and transport models (CTMs). Here, we present leaf-scale measurements of $\chi_s$ for hybrid poplar clone '546' (Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) growing in two N treatments (N0, no N added; N50, 50 kg N ha$^{-1}$ yr$^{-1}$ urea fertilizer added) and two O$_3$ treatments (CF, charcoal-filtered air; E-O$_3$, non-filtered air plus 40 ppb) for 105 days. Our results showed that $\chi_s$ was significantly reduced by E-O$_3$ (41%) and elevated N (19%). The interaction of N and O$_3$ were significant, and N can mitigate the negative effects of O$_3$ on $\chi_s$. Elevated O$_3$ significantly reduced the light-saturated photosynthetic rate ($A_{sat}$) and chlorophyll (Chl) content and significantly increased intercellular CO$_2$ concentrations (Ci), but had no significant effect on stomatal conductance ($g_s$). By contrast, elevated N did not significantly affect all measured photosynthetic parameters. Overall, $\chi_s$ was significantly and positively correlated with $A_{sat}$, $g_s$ and Chl, whereas a significant and negative relationship was observed between $\chi_s$ and Ci. Our results suggest that O$_3$-induced changes in $A_{sat}$, Ci and Chl may affect $\chi_s$. Our findings provide a scientific basis for optimizing parameterizations of $\chi_s$ in atmospheric chemistry and transport models to respond to environmental change factors (i.e., elevated N deposition and/or O$_3$) in the future.

**Keywords:** Ammonia; Ozone; Apoplast; Compensation point; Forest species

**Capsule:** Both elevated O$_3$ and fertilization can significantly reduced NH$_3$ stomatal compensation point ($\chi_s$) of poplar clone '546', and fertilization can mitigate the negative effects of O$_3$ on $\chi_s$.

## 1. Introduction

Atmospheric ammonia (NH$_3$) is the primary alkaline trace gas in the atmosphere and plays a vital role in many biogeochemical and atmospheric processes (Behera et al., 2013). It neutralizes atmospheric acids to yield ammonium (NH$_4^+$) aerosols,
which results in increased mass loadings of fine atmospheric particulate matter (PM<sub>2.5</sub>, aerodynamic diameter ≤ 2.5) (Xu et al., 2016, 2017), thereby reducing visibility and adversely impacting ecosystem and human health (Gu et al., 2014). By contrast, atmospheric deposition of reduced N (NH<sub>3</sub> and NH<sub>4</sub><sup>+</sup>) can cause soil acidification (Du et al., 2015), eutrophication (Pareman et al., 2016) and loss of biodiversity (Erisman et al., 2007) in sensitive ecosystems.

Plants can be either a source or a sink of atmospheric NH₃, depending on the difference between atmospheric NH₃ concentration and the so-called canopy NH₃ compensation point (Massad et al., 2010). As a major component of canopy NH₃ compensation point, ammonia stomatal compensation point (χs) is defined as the atmospheric NH₃ concentration for which there is no exchange between the leaf and the atmosphere under dry conditions (Flechard et al., 2013). Theoretically, χs is also the gaseous NH₃ concentration in the leaf sub-stomatal cavity that is in equilibrium with ammonium concentration in the apoplast (Husted and Schjoeering, 1995). It plays a vital role in controlling the magnitude and the direction of NH₃ exchange between the canopy and the atmosphere (Sutton et al., 1995). Specifically, if atmospheric NH₃ concentrations exceed χs then NH₃ deposition from the atmosphere to vegetation will occur, while with atmospheric NH₃ concentrations below χs, there will be a net emission of NH₃ by plants. χs depends directly on the plant nitrogen (N) status, developmental stage, and environmental conditions (e.g., N fertilization), with larger values generally observed under conditions of high N supply to the soil-plant system and at senescence (Massad et al., 2009; Schjoerring et al., 1998).

χs can be derived from simultaneous measurement of vertical fluxes and concentrations of NH₃ by using micrometeorological flux techniques over large fields (Hansen et al., 2017; Nemitz et al., 2001; Personne et al., 2015), or in chambers by finding the concentration at which the total flux is zero (Hill et al., 2001; Massad et al., 2009; Wang et al., 2011). In addition, the bioassay approach has also been developed for assessing χs and it is based on the determination of the leaf apoplastic NH₄⁺ concentration and pH by mean of apoplast extraction (Husted and Schjørring, 1995). These two methods are complementary. Apoplast extraction is more appropriate for leaf
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and cell scale processes whereas chamber/micrometeorological measurements tend to be more appropriate for flux measurements at an entire plant/canopy scale (Massad et al., 2009; Sutton et al., 2009).

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Forests represent a major uncertainty in quantification of regional NH₃ fluxes and parameterization of bi-directional NH₃ exchange in atmospheric chemistry and transport models (CTMs) such as AURAMS (A Unified Regional Air-quality Modelling System, Zhang et al., 2010) and CMAQ (Community Multiscale Air-Quality Modeling System, Fu et al., 2015). This is not only due to the large land area of forests but also because of the wide range of forest types and management practices. In conditions of bi-directional NH₃ exchange, forests are of particular interest. For example, temperate deciduous forests are potentially a natural source of NH₃ (Hansen et al., 2013, 2017; Neirynck and Ceulemans, 2008), leading to impact of forests on the atmospheric NH₃ level. In contrast, tropical humid forest and temperate coniferous forest can acts as net NH₃ sinks (Bertolini et al., 2016; Duyzer et al., 2005), resulting in the impact of atmospheric NH₃ on the ecological functioning of forests.

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$\chi_s$ is one of the key parameters for parameterizations of NH₃ incorporated into CTMs (Massad et al., 2010). Based on published data on $\chi_s$ in relation to different plant species, growth stages, N supply etc., Massad et al. (2010) derived a new operational parameterization for integrating bi-directional NH₃ exchange into CTMs, However, uncertainties still exist for its parameterization, partially due to the following two drawbacks: 1) measurement of $\chi_s$ for different ecosystems, specific to forests, is very sparse and is only considered for a single growth stage of plants; 2) the relationships established between N fertilizer application and $\chi_s$ remain uncertain due to a lack of co-measurement of $\chi_s$ with different organic fertilizer (manure, slurry and urea) application rates. In addition, the actual parameterization of NH₃ exchange models requires large databases accounting for the variability of $\chi_s$. To our knowledge, there is only one process-based model developed by Riedo et al. (2002) for grasslands which accounts for the plants N nutrition and growth stage in calculating $\chi_s$. However, as $\chi_s$ is not only driven by N input to the ecosystem and plant growth stage, it may be a strongly regulated process that depends on environmental changes such as elevated ground-level
110 O₃.

111 Ground-level O₃ can be considered as the most phytotoxic air pollutant due to
112 visible injury to a variety of plants and the rising concentrations in different regions of
113 the world (Cooper et al., 2014; Feng et al., 2014). It affects photosynthetic parameters
114 (e.g., stomatal conductance (g_s), light-saturated CO₂ assimilation rate (A sat),
115 intercellular CO₂ concentration (Ci) and chlorophyll (Chl) content) of forest species to
116 a varying extent (Li et al., 2017). In contrast, atmospheric N deposition represents an
117 important nutrient from the environment for plants (Liu et al., 2010). In N-limited
118 ecosystems (e.g., forest) N deposition might enhance photosynthetic activity (i.e.
119 photosynthetic enzyme activity) and net primary productivity (N fertilization effect)
120 (Liu et al., 2011). In the context of an N-saturation ecosystem, however, N deposition
121 may render plants more susceptible to pollutants and natural environmental stressors
122 (Cardoso-Vilhena and Barnes, 2001). Ozone and N induced changes in the growth and
123 metabolism of plants may affect the χs of plants due to a clear link between χs and
124 photosynthetic parameters. For example, Mattssone and Schjoerring (1996) showed
125 that leaf NH₃ emission from Hordeum vulgare L. cv. Golf plants showed a consistent
126 diurnal pattern with photosynthesis but the opposite trend with g_s. Furthermore,
127 Schjoerring et al. (1998) found that NH₃ emission from leaves of Brassica napus L.
128 plants increased with Chl degradation. Such results demonstrate that there are
129 corresponding influences of those parameters on χs, which positively impacts leaf NH₃
130 emission (Massad et al., 2010). In this context, understanding the effects of elevated O₃
131 and N as well as their influence on the plant physiological parameters controlling χs is
132 important for prediction of χs. Unfortunately, the relevant information for different
133 forest species is still unknown, significantly restricting the optimization of the χs
134 parameter in CTM models.

135 Poplars are widespread deciduous plants in temperate and boreal forests. In China,
136 poplar is a native species, with a cultivated area of more than 10 million ha (Yuan et al.,
137 2016). We designed an experiment to investigate for the first time the individual effects
138 of elevated N deposition (with controlled application of urea) and O3 and their
139 interactions on χs of hybrid poplar clone ‘546’ (Populus deltoides cv. 55/56 x P. deltoides
cv. Imperial). In addition, we estimated the relationships between photosynthetic parameters ($g_s$, $A_{sat}$, Ci and Chl) and $\chi_s$, and discuss how N and $O_3$, as well as their-driven modifications in the aforementioned photosynthetic parameters, respectively affect $\chi_s$.

## 2. Materials and methods

### 2.1. Experimental site and plant materials

The study was conducted in Yanqing Field and Experimental Basin, Tangjiapu village, Yanqing District (40°29′N, 115°59′E, 500 m.a.s.l.), about 74 km northwest of Beijing city centre. When the winds come from the north or northwest, this basin is located upwind of the Beijing urban area. The site is characterized by a continental monsoon climate, with mean annual temperature of 9 °C and mean annual precipitation of 400-500 mm.

Rooted cuttings of hybrid poplar clone '546' (Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were planted on 7 May 2017 and cultivated in individual 20 L circular plastic pots when they were about 31 days old. The plots were filled with local light loamy farmland soil, which was excavated at 0-10 cm depth, sieved out by a 0.3 mm pore mesh and then thoroughly mixed for homogeneity. Plants with similar height (ca. 27.2 cm) and basal stem diameter (ca. 4.5 mm) were selected and pre-adapted to open-top chambers (OTCs, octagonal base, 12.5 m² of growth space and 3.0 m height, covered with toughened glass) for 10 days before $O_3$ fumigation. All seedlings were manually irrigated at 1-2 day intervals in order to keep moisture at a similar level to that in farm fields.

### 2.2. *O*₃ and *N* treatments

The experiment was conducted in six OTCs with two $O_3$ treatments: charcoal-filtered ambient air (CF), and elevated $O_3$ (E-O₃, non-filtered air with targeted $O_3$ addition of 40 ppb during fumigation). Each treatment had three OTC replicates, and six potted plants were randomly distributed in each OTC. The $O_3$ fumigation was performed from 10 June to 22 September 2017. The daily fumigation time was 10 h (from 08:00 till 18:00), except on rainy days. During the fumigation period, the
averaged O₃ concentrations in CF and E-O₃ were 24.0 and 80.6 ppb, respectively, and AOT40 (accumulated hourly O₃ concentration above a threshold of 40 ppb) was 2.4 and 41.6 ppm h, respectively. Ozone was generated from pure oxygen using an electrical discharge O₃ generator (HY003, Chuangcheng Co., Jinan, China) and mixed with ambient air using a fan. Ozone concentrations inside the OTCs were continuously monitored at approximately 10 cm above the plant canopy using two ultraviolet (UV) absorption O₃ analyzers (Model 49i-Thermo, Thermo Scientific, Massachusetts, USA, one for three OTCs). The analyzers were calibrated with a 49iPS calibrator (Thermo Scientific) before the experiment and once a month during the experiment.

In addition to the O₃ treatments, two N treatments were applied with three replicates: control (N0, no N added), and moderate N, (N50, 50 kg N ha⁻¹ yr⁻¹). For N50, N additions were applied five times (13 June, 29 June, 16 July, 1 August, 17 August) to the soil with dilute urea solutions by using a 50 mL plastic bottle and the control pots received equal amounts of pure water. In total, N50 received 0.245 g N per pot (i.e. 0.526 g urea per pot) throughout the experiment. This application rate (0.245 g N per pot) can be projected to a dose of 50 kg N ha⁻¹ based on the area (0.049 m²) of each pot. The concentrations of NH₄⁺-N and NO₃⁻-N in the soil of N0 treatment were 6.5 ± 2.2 and 4.6 ± 1.4 mg N kg⁻¹ wet soil, respectively (unpublished data).

## 2.3. *Measurements of physiological parameters*

Measurements of gas exchange, leaf temperature and chlorophyll content were performed during two periods, i.e., at the end of July and August, 2017 (on 30 and 31 July, and on 29 and 31 August, respectively). For all plants, middle leaves were selected as targeted leaves, which were 11th to 13th fully expanded leaves from the apex and comprised the main part of the leaves on each plant. A portable photosynthetic system fitting with a 6400-40 leaf chamber fluorometer (LI-6400-40, LI-COR Co., USA) was used to measure gas exchange and leaf temperature from one middle leaf between 9:00 and 11:00 h. For the measurements, the photosynthetic photon flux density was set at 1200 µmol m⁻² s⁻¹, the CO₂ concentration of air entering the leaf at 400 µmol mol⁻¹ and the relative humidity at 50-60%. The measured parameters were Aₜ sat, gs, and Cₜ. It
should be noted that the 2-h measurement duration has only a small influence on g<sub>s</sub>, since the influencing factors on g<sub>s</sub> (e.g., leaf temperature, vapor pressure deficit, ambient CO<sub>2</sub> concentration, Hu et al., 2015) have a small fluctuation during the corresponding time period (data not shown), and the photosynthetic photon flux density leaf was fixed. During the entire experimental period, a total of 72 leaf samples were measured.

Immediately after measurements of gas exchange and leaf temperature, two leaf discs were sampled from the targeted leaf and then extracted with 2 mL 95% ethanol solution in the dark for at least 72 h at 4 °C. The Chl content in the extract was determined using the specific absorption coefficients (Lichtenthaler, 1987).

## 2.4. Determination of apoplastic NH<sub>4</sub><sup>+</sup> and H<sup>+</sup> concentration

A slightly modified version of the vacuum infiltration technique developed by Husted and Schjoerring (1995) was employed to determine apoplastic NH<sub>4</sub><sup>+</sup> and H<sup>+</sup> concentrations. Immediately after measurements of physiological parameters, the targeted leaf was cut and washed with high-purity water (18.2 Ω), in order to avoid any contamination from air pollutants (e.g., particulate NH<sub>4</sub><sup>+</sup>). The leaf was then blotted dry with clean absorbent paper towel and the central petiole was removed. The leaves were separated into three replicates and then weighted, infiltrated with 280 mM sorbitol solution using a 60 mL plastic syringe with a series of vacuum/pressure for 5 min. The vacuum/pressure process was automatically applied with an intercellular fluid extractor (NS-AFE-1, Pulanta Co. Suzhou, China). The infiltrated leaves were quickly rinsed with high-purity water, blotted dry and re-weighted. The leaves were then rolled, inserted into tubes and centrifuged at 9000 r min<sup>-1</sup> for 10 min at 4°C to collect the apoplastic solution. Cytoplasmic contamination of the apoplast during the extraction procedure was checked by performing the extraction using a buffered solution (0.1 M Ntris[hydroxymethyl]methyl-2-aminoethanesulphonic acid, 2 mM dithiothreitol and 0.2 mM EDTA), and comparing the activity of Malate Dehydrogenase (MDH) in the apoplastic extracts to its activity in bulk tissue extracts as described by Husted and Schjørring (1995). The contamination was less than 1.2 ± 1.1% of MDH activity in
227 apoplast extract relative to bulk tissue extract. The extracted solution was then frozen
228 and stored at -20 °C prior to chemical analysis.

229 The NH₄⁺ concentrations in the apoplastic extracts were measured with an AA3
230 continuous-flow analyzer (BranCLuebbe GmbH, Norderstedt, Germany). The
231 detection limit of NH₄⁺ was 0.01 mg N L⁻¹. The pH of the extracted solution was
232 measured with an InLab micro electrode (Mettler Toledo, Udorf, Switzerland). The
233 dilution of the apoplastic solution was determined spectrophotometrically at
234 wavelength 492 nm for the sorbitol, which allowed the calculation of a dilution factor
235 (Hove et al., 2002). The concentration of apoplastic NH₄⁺ and H⁺ was corrected for
236 dilution during the extraction procedure by multiplication with the dilution factor (F₂dil).
237

238 The aqueous volume of the apoplast (Vₐpo, mL g⁻¹ leaf fresh weight (LFW)) was
239 estimated using the equation (Hove et al., 2002):

$$
239 \qquad V_{\text{apo}} = \frac{V_i(F_{\text{d,sorb}} - 1)}{\text{LFW}} \qquad (1)
$$

240 where Vᵢ is the infiltration volume which was calculated by assuming a leaf density
241 of 1 g cm⁻³ the difference in weight before and after infiltration, Fd_sorb is the dilution
242 factor of sorbitol.

$$
243 \qquad F_{\text{dil}} = \frac{V_{\text{apo}} + V_{\text{air}}}{V_{\text{apo}}} \qquad (2)
$$

244 where V<sub>air</sub> is the air volume inside the leaf (cm<sup>3</sup> g<sup>-1</sup> LFW) which was measured by
245 infiltrating leaves with low-viscosity silicone oil (10 mPa s). Based on the increase in
246 weight and oil density (0.93 g cm<sup>-3</sup>), the V<sub>air</sub> was estimated to be approximately 0.16
247 mL g<sup>-1</sup> throughout the experiment.

248
2.5. Determination of the leaf tissue NH₄⁺ concentration

249 The leaf segments were cut into small pieces, frozen in a ceramic mortar with
250 liquid N (-210 °C), and quickly ground into a homogenous powder using a ceramic
251 pestle. The weighed samples (approximately 1.0 g per sample) were put into a 5 mL
252 centrifuge tube with 4 mL of high-purity water, followed by centrifugation at 2000 g
253 (4°C) for 10 min (Loubet et al., 2002). The supernatant was then decanted and filtered
254 through a syringe filter (0.45 µm, Tengda Inc., Tianjin, China) to remove large plant
tissues. The filtered solution was frozen and stored at -20 °C until analysis for NH₄⁺ using an AA3 continuous-flow analyzer as mentioned before.

## 2.6. Calculation of NH₃ stomatal compensation point

The stomatal compensation points were derived using the apoplast pH and NH₄⁺ concentrations (Loubet et al., 2002) according to the equation:

$$ \chi_s = M_{NH_3} \times K_H \times K_D \times \frac{[NH_4^+]}{[H^+]} \times 10^9 \quad (3) $$

where χs is the stomatal compensation point in µg NH₃ m⁻³, MNH₃ is the molecular mass of NH₃ in g mol⁻¹, The ratio of apoplast NH₄⁺ to apoplast H⁺ concentration, called the emission potential (expressed as Γs), is temperature independent and dimensionless (Massad et al., 2010). KH is the Henry constant and KD is the dissociation constant. The product KHKD depends on temperature and was calculated following the method of (Hill et al., 2001):

$$ K_H K_D = \frac{161512}{T_{leaf}} \times 10^{\frac{-4507.11}{T_{leaf}}} \quad (4) $$

where Tleaf is leaf temperature in Kelvin.

## 2.7. Statistical analysis

Data of each investigated variable from three plants per OTC were averaged and then used as the statistical unit (N=3). Prior to analysis, all data were tested for normality using the Shapiro-Wilk's W-test and for homogeneity of variance using Levene's-test to determine whether data should be transformed to satisfy statistical assumptions. A split-plot design was used, with O₃ as the main plot, N level as sub-plot and measurement dates as repeat-measurement. A repeated measure analysis of variance (ANOVA) with a mixed linear model was then conducted to examine the effects of O₃, N, measurement dates and their interactions on physiological parameters and χs as well as other parameters. Tukey's Honestly Significant Difference (HSD) test was applied to examine the significant differences. Analysis of covariance (ANCOVA) was performed to test the significance of difference in the slopes of the linear relationship between χs and physiological parameters. All statistical analyses were performed in R version 3.3.3 (http://www.r-project.org/), with significant level set at
P<0.05. All the data were shown as mean ± standard deviation (SD) of three OTC replicates.

## 3. Results

For all investigated variables, similar and non-significant responses to E-O₃ and N50 were found between the two measurement dates, i.e., July and August (Figs. 1 and 2). Based on integrated analysis of all data from all N and O₃ treatments and the two measurement dates, the main results are presented below.

### 3.1. Photosynthetic parameters

For all investigated variables ($A_{sat}$, $g_s$, Ci and Chl), the interactions of O₃ and N were not significant, but the individual effects of them were significant for most variables (Table 1). Relative to CF, E-O₃ significantly reduced $A_{sat}$ by 55%, whereas $A_{sat}$ has a small non-significant increase (6%) in NF50 relative to N0 (Fig. 1a, Table 1). $A_{sat}$ significantly decreased (by 24%) in August relative to that in July, when averaged across all treatments. The effects of N and O₃ on $g_s$ were both not significant, whereas measurement date significantly affected $g_s$ (Fig. 1b, Table 1). Similar to $A_{sat}$, $g_s$ significantly decreased by 30% in August compared with that in July.

Ozone significantly influenced Ci and Chl, whereas N had no significant effects on them (Fig. 1c d, Table 1). Ci was significantly increased by E-O₃ (+7%) compared with CF, and also was significantly higher (6%) in August than in July. Conversely, Chl was significantly reduced by E-O₃ (-30%), and significantly decreased (by 57%) in August.

### 3.2. Leaf apoplastic NH₄⁺ and pH, and leaf tissue NH₄⁺

Fig. 2a-c shows the responses of leaf apoplastic NH₄⁺ concentration and pH, and leaf tissue NH₄⁺ concentration to E-O₃ and N50. The interactions between O₃ and N and/or measurement dates were not significant for all those three variables (Table 1). However, individual effects of them on apoplastic NH₄⁺ concentrations and pH all reached statistically significant levels. Similarly, leaf tissue NH₄⁺ concentration was significantly influenced by both O₃ and measurement dates, but not by N.

Averaged apoplastic NH₄⁺ concentration was significantly reduced (by 15%) in E-
O₃ plants compared with CF plants (Fig. 2a, Table 1). By contrast, the mean concentration was significantly increased (by 27%) in N50 plants compared with that in N0 plants. A significant increase (14%) of the mean occurred in August relative to July. As for apoplastic pH, two small but significant reductions (both 4%) of the mean were found in E-O₃ and N50 plants compared with those in CF and N50 plants, respectively (Fig. 2c, Table 1). Also, the mean pH was significantly lower (15%) in plants grown in August than in July.

In contrast to apoplastic NH₄⁺, the mean tissue NH₄⁺ concentration significantly increased (by 15%) in E-O₃ plants relative to that in CF plants, whereas a small and non-significant difference in the mean was found between N0 and N50 plants (Fig. 2b, Table 1). The mean concentrations decreased significantly (on average by 6%) in August relative to July.

### 3.3. Emission potential ($\Gamma_s$) and stomatal compensation point ($\chi_s$)

As presented in Table 1, both O₃ and measurement dates significantly affected $\Gamma_s$, whereas N had no significant effect on it. Also, no significant interactions between O₃, N and/or measurement dates were observed. E-O₃ treatment relative to CF significantly reduced $\Gamma_s$ by 39% (Fig. 2d). $\Gamma_s$ was significantly reduced (by 85%) in August compared with that in July.

$\chi_s$ was significantly lower in E-O₃ than in CF (0.29 ± 0.29 and 0.48 ± 0.45 µg NH₃ m⁻³), and also in N50 than N0 (0.34 ± 0.36 and 0.42 ± 0.41 µg NH₃ m⁻³) (Fig. 3, Table 1). A significant interaction of O₃ and N was found (Table 1). $\chi_s$ was significantly lower in August than in July (0.07 ± 0.05 and 0.70 ± 0.29 µg NH₃ m⁻³) (Fig. 3, Table 1).

### 3.4. Correlations between stomatal compensation point ($\chi_s$) and photosynthetic parameters

$\chi_s$ was positively and significantly correlated with $A_{sat}$ and $g_s$ (Fig. 4a, b), whereas a negative and significant correlation between $\chi_s$ and $C_i$ was observed (Fig. 4c). The correlation between $\chi_s$ and $C_i$ was positive and significant in July, but was not significant in August (Fig. 4d).

## 4. Discussion

### 4.1. Effect of N application
The application rate of urea fertilizer in the present experiment (50 kg N ha⁻¹ yr⁻¹) is approximately 2.3 and 3 times higher than the averages of N deposition over China (16 kg N ha⁻¹ yr⁻¹ in 2008-2012 period, Zhao et al., 2017) and in China's forests (22 kg N ha⁻¹ yr⁻¹ in 1995-2010 period, Du et al., 2014), respectively. It is also approximately 1.7-5.0 times greater than reported values during recent years in the N deposition hotspots of western Europe (20.0 to 28.1 kg N ha⁻¹ yr⁻¹, Vet et al., 2014), and North America (10.0 to 20.0 kg N ha⁻¹ yr⁻¹, Li et al., 2016). According to Liu et al. (2013), N deposition increased by approximately 8 kg N ha⁻¹ yr⁻¹ between the 1980s and the 2000s in China. Also, total N deposition is expected to have a 5-10% increase in the year 2050 relative to 2005 (Kanakidou et al., 2016). In view of the above, the level of N addition in this study is sufficient to assess the ecological effects of enhanced N deposition expected in the future. However, it should be pointed out that application of N to the soil is not able to fairly reflect the process of N deposition in the real-world, in which natural deposited N would at least partially be captured by the canopy. As reported by Du et al. (2014), for example, canopy captured dry deposition of total inorganic N in China's forest was estimated at 6.9 kg N ha⁻¹ yr⁻¹.

Apoplastic NH₄⁺ concentration is one of the key factors in controlling χₛ, which is associated with the status of leaf N and external N supply (e.g., N fertilization and atmospheric deposition) (Herrmann et al., 2009; Massad et al., 2008). The apoplastic NH₄⁺ concentrations measured for the N0 and N50 treatments (but without E-O₃) were 0.10 ± 0.01 and 0.13 ± 0.01 mM, respectively (Fig. 2a). To our knowledge published data on apoplastic NH₄⁺ concentrations in leaves of poplar clone 546 is unavailable, making a direct comparison with other studies impossible. However, our measured value for N0 treatments were close to that reported for *Fagus sylvatica* growing in July and August (0.11 mM, Wang et al., 2011). Compared with agricultural crops, our measured value for N0 treatments was higher than that reported for barley (0.04 mM, Mattsson et al., 1998), and was close to the value of 0.10 measured for oilseed rape (Massad et al., 2009) both growing on N0. The value for plants grown on N50 was similar to that measured for oilseed rape (0.18 mM with 6 mM NO₃⁻, Schjoerring et al., 2002), and was lower than the value of 1.9 mM observed for barley grown on 5 mM
372 NH₄⁺ (Mattsson et al., 1998). As expected, enhanced N input (N50) significantly increased the apoplastic NH₄⁺ concentration (Fig. 2, Table 1), probably due to increased soil N availability. This result is similar to the findings of Massad et al. (2009) that the NH₄⁺ concentrations in leaf apoplast of oilseed rape increased significantly with rising N treatments (aside from NO₃⁻ supply).

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Besides apoplastic NH₄⁺ concentration, the pH of the apoplast may be the most important factor determining χs. There is evidence that NH₄⁺-fed plants have reduced apoplastic pH compared to NO₃⁻-fed plants, e.g., sunflower (Hoffmann et al., 1992), soybean (Kosegarten and Englisch, 1994) and barley (Mattsson et al., 1998), probably due to a root rather than shoot assimilation of N (Pearson et al., 1998). Application of urea fertilizer in the present experiment probably enhanced soil NH₄⁺ level via urea hydrolysis, which further significantly increased apoplastic NH₄⁺ concentrations (Fig. 2a) via root assimilation and the subsequent transport of NH₄⁺ to the foliar apoplast in the xylem (Mattson et al., 1998). This behaviour, along with NH₄⁺ uptake-induced acidification may offer an explanation for lower apoplastic pH (ranging from 4.6 to 6.0, Fig. 2c) of poplar 546 compared with those reported for most plant species (between 5.0 and 6.5, Grignon and Sentenac, 1991). However, it is still a little controversial how the type of mineral N supply (NO₃⁻ versus NH₄⁺) affects apoplastic pH in leaves. Nitrate type mineral N (NO₃⁻) has been shown to an alkalization while NH₄⁺ induced an acidification (Hoffmann et al., 1992; Mengel et al., 1994). Sattelmacher. (2001) suggested that nitrate effectively leads to higher pH levels but only depending on its concentration in the xylem sap. However, NH₄⁺ nutrition didn't show any effect on leaf apoplast pH at low concentrations. Finnemann and Schjoerring (1999) questioned this result and show a relation between root N supply type and NH₄⁺ concentrations in the xylem sap and in the leaf apoplast. They explain this result by high levels of photorespiration (Kronzucker et al., 1998; Nielsen and Schjoerring, 1998). However there is a clear effect on apoplastic pH due to foliar application of NH₄⁺ which leads to lowed pH values (Peuke et al., 1998). On the other hand, fumigation with NH₃ decreases H⁺ activity (Hanstein et al., 1999).

401 Enhanced N input significantly increased both apoplastic NH₄⁺ and H⁺
concentration with a similar significant level (Figs. 2a,c and Table 1). Conversely, χs
was significantly reduced (by 19%) by N50 (Fig. 3). These results, together with the
relatively low calculated χs throughout, especially in August (Fig. 3), suggest that χs
appears to be pH-driven in this study. The apoplastic pH of Phaseolus vulgaris has been
found to become alkalinized during photosynthesis (Raven and Farquhar, 1989).
Similarly, we found that the N load marginally significantly (P=0.057) increased Asat of
poplar 546 (Fig. 1a, Table 1), which significantly positively correlated with apoplastic
pH (Fig. 5a) but had no significant relationship with apoplastic NH4+ concentration
(R²=0.09, P=0.141). These results provide an explanation for a significant and positive
relationship observed between Asat and χs (Fig. 4a). Although such an increase in
apoplastic pH occurred due to non-significant increased Asat by N load, it is insufficient
to offset NH4+ uptake-induced apoplast acidification, leading to relatively low χs.

4.2. *Effect of O*<sub>3</sub> *application*

According to monitoring results for the 2014-2016 period in 187 Chinese cities,
the mean daily 8-h O₃ concentrations peak in summer reached up to 114.30 ± 23.78 ppb
(Li et al., 2017). Obviously, the O₃ level in E-O₃ was within the range of current O₃
levels in China and was also in the range of future expected concentrations in warm and
sunny areas of the world at the end of this century (The Royal Society 2008; IPCC
2013). The dose of O₃ (41.6 ppm h in AOT40) in E-O₃ by far exceeded the O₃ exposure
limit of 5 ppm h accumulated over the growing season for forest protection (CLRTAP,
2015) and 12 ppm h for poplar protection (Hu et al., 2015). As anticipated, O₃-induced
injuries to poplar 546 were detected in E-O₃ despite N fertilization (e.g., reduction of
Asat and Chl, Fig. 1a, d), thus confirming that poplar 546 is a very sensitive species to
O₃ (Shang et al., 2017).

E-O₃ significantly increased Ci and decreased Asat and Chl, respectively, but did
not significantly affect gs (Figs. 1 and Table 1). These results are consistent with the
findings of Shang et al. (2017) for polar clone 546. Similarly, a previous study showed
that O₃ can substantially reduce Asat in most plants, and also detected an uncoupled
relationships between Asat and gs (Zhang et al., 2012). This can be explained by the fact
that the O₃-induced reduction in Asat is largely ascribed to non-stomatal factors, i.e.
432 impaired physiological activity of mesophyll cells (Akhtar et al., 2010; Feng et al., 2016). Although each investigated photosynthetic parameter was significantly influenced by O₃, interactions of O₃ and N were not significant (Table 1). This is similar to the findings of Harmens et al. (2017) that no any interactions between O₃ and N regarding photosynthetic parameters, chlorophyll content, gs, N content in senesced leaves and leaf number for *Betula pendula* saplings.

438 We found that E-O₃ significantly reduced the calculated χs (Fig. 3). This is most 439 likely related to a decline in apoplastic pH resulting from O₃-induced changes in 440 photosynthetic parameters, i.e., decreased Asat and Chl, and increased Ci (Fig. 1a,c,d, 441 Table 1). This is because in addition to Asat, both Chl and Ci correlated significantly 442 with χs and apoplastic pH (Figs. 4c,d and 5b,c). In addition, there was a large positive 443 response of ammonia emission (from young barely) to photosynthesis (Mattsson and 444 Schjoerring, 1996), mainly due to the fact that NH₃ assimilation by plants requires 445 carbon skeletons generated from photosynthesized carbohydrates for the synthesis of 446 amino acids (Huppe and Turpin, 1994). In this regard, a significant reduction in Asat can 447 also give rise to declines in apoplastic NH₄⁺ concentration. Similar to N, ozone has a 448 greater impact on apoplastic pH than on apoplastic NH₄⁺ concentration, as 449 demonstrated by the significant reduction of Γs by O₃ (Fig. 2d, Table 1); this therefore 450 led to a significant reduction in χs (Fig. 3, Table 1).

451 **4.3. Combined effects of N and O₃ application**

452 The present results showed that N and O₃ interacted significantly in χs (Table 1).
453 Further analysis showed that χs in N0*E-O₃ (0.28 ± 0.30 µg NH₃ m⁻³) was 50% lower
454 than that in N0*CF (0.56 ± 0.49 µg NH₃ m⁻³), suggesting a negative effect of E-O₃ on
455 χs. This, together with a 28% decrease in χs when comparing χs in N50*E-O₃ (0.29 ±
456 0.31 µg NH₃ m⁻³) with that in N50*CF (0.40 ± 0.44 µg NH₃ m⁻³), also indicate that N
457 application can mitigate the negative effects of O₃ on χs.

458 Our selected poplar 546 belongs to deciduous broadleaf species. To make a 459 preliminary assessment of whether Chinese deciduous broadleaf forests (Fig. 6a) act as 460 a source or a sink for atmospheric NH3, we compared the calculated χs with mean 461 atmospheric NH3 concentration in July (Fig. 6b) and August (Fig. 6c) during 2008-
2012 period. The concentrations were modeled using the GEOS-Chem model and fitted
with the surface NH₃ measurements, see details in Zhao et al. (2017)). The results of
the comparison show that the modeled NH₃ concentrations over approximately 91%
and 100% of total land area exceeded the calculated χ₃ for elevated N and O₃ treatment
in July (average 0.54 µg NH₃ m⁻³) and August (average 0.03 µg NH₃ m⁻³), respectively.
It should be noted that such percentages are considered to be approximate estimates, as
χ₃ may vary among different forest species, and the current atmospheric NH₃
concentration cannot represent future NH₃ levels. Nevertheless, we may conclude that
under the current ambient NH₃ concentrations in China the canopy of deciduous
broadleaf forests is unlikely to be a major source of NH₃ emission during summertime.

472

4.4. *Dependence on measurement date*

χ₃ can be influenced by plant developmental stage since leaf apoplastic NH₄⁺
concentration and pH varies among leaves of different ages (Hill et al., 2002). Mattsson
and Schjoerring (2003) reported that, for ryegrass (Lolium perenne), both apoplastic
and tissue NH₄⁺ concentrations were 2-3 times higher in senescing or injured leaves
(with visual symptoms of senescence) compared with green leaves. The present study
shows that leaf apoplastic NH₄⁺ concentrations significantly increased in August
compared with those in July, whereas a significant reduction was found for leaf tissue
NH₄⁺ concentration (Fig. 2a, b). In addition, a negative relationship was observed
between NH₄⁺ concentrations in apoplast and in tissue (Fig. 5d). These results together
indicated that NH₄⁺ is actively transported from the leaf tissue to the apoplast. This
explanation is supported by evidences from earlier studies showing that apoplastic fluid
in leaves constitutes a highly dynamic NH₄⁺ pool, to which NH₄⁺ is constantly supplied
via NH₃ efflux from the mesophyll cells (Nielsen and Schjoerring, 1998; Schjoerring et
al. 2000).

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Regarding apoplastic pH, the values measured in August were significantly
reduced compared to July, and almost all were <5 (Fig. 2c, Table 1), for which an
explanation is the combined effect of NH₄⁺ uptake-induced acidification (see Sect. 4.1)
and O₃-accelerated leaf senescence (Gao et al., 2017). As reported by Mattsson and
Schjoerring (2003), leaf aging from green leaves to yellow tips resulted in a pronounced decrease of pH by more than 1 unit. Thus, a significantly lower Asat in August resulting from accelerated leaf senescence (Fig. 1a, Table 1) also contributed to lower apoplastic pH due to the existence of apoplast alkalinization during photosynthesis as mentioned earlier.

$\chi_s$ was significantly reduced in August compared with July (Fig. 3). This is likely due to the greater impact of measurement date on apoplastic pH than on apoplastic $NH_4^+$ concentration as indicated by their respective significant level (Table 1). The change in stomatal opening is an important control mechanism for the regulation of influxes and outfluxes of $NH_3$ into or out of the leaves because the conductance for diffusion of $NH_3$ is affected (Schjoerring et al., 1998). We found that $g_s$ significantly decreased in August (Fig. 1b, Table 1). Also, a positive and significant relationship was observed between $g_s$ and $\chi_s$ (Fig. 4b); this is mainly caused by measurement date as both E-O$_3$ and N did not significantly affect $g_s$. These results together suggested that a significant reduction of $g_s$ also contributed to a reduction in $\chi_s$.

### 4.5. Uncertainty and recommendations

In the present study, $\chi_s$ is calculated based on direct measurements of leaf apoplastic $NH_4^+$ concentration and pH by means of extraction of the apoplastic fluid with successive vacuum infiltration/centrifugation technique (Husted and Schjørring, 1995). Although this technique has been successfully applied to several plant species in the field (Herrmann et al., 2009; Mattsson et al., 2009), it is subject to uncertainties regarding potential regulation of apoplastic $NH_4^+$ concentration and pH by the plant during the infiltration and buffering effects (Massad et al., 2009). For example, Nielsen and Schjørring (1998) showed that apoplastic $NH_4^+$ concentration in *Brassica napus* L. appeared to be regulated during infiltration. However, Hill et al. (2001) did not detect such a homeostasis for apoplastic $NH_4^+$ concentration in *Luzula sylvatica* (Huds.) Gaud. Following the method of van Hove et al. (2002), $F_{dil}$, calculated based on determination of $V_{air}$ and $V_{apo}$ (Equ. 2), was applied to correct apoplastic concentrations. $V_{apo}$ values obtained in the present work varied from 0.06 to 0.23. These values fit well into the range found by other researchers for different plant species (Van Han et al., 2001, 2002).
The measured $V_{\text{air}}$ (0.16 ml g⁻¹ LFW) was also comparable to values (0.21 mL g⁻¹ LFW) reported for *Lolium perenne* L. (Van Han et al., 2002). These results indicate that the value for $F_{\text{dil}}$ determined in the present study was acceptable. However, due to a lack of information regarding infiltration of poplar 546, use of $F_{\text{dil}}$ might also result in some uncertainties in apoplastic $NH_4^+$ concentrations and pH, and probably in $\chi_s$ if there is a potential difference in the buffering capacity between them.

The vacuum infiltration/centrifugation method is also subject to some uncertainty due to the strong possibility of cytoplasmic contamination of the apoplast during the extraction procedure (Lohaus et al., 2001). We estimated the error in this method by assaying the contamination of the apoplast by MDH activity. The cytoplasmic contamination in the present work was about 1.2%.

$\chi_s$ obtained in the current study was calculated at the leaf scale and only for the middle leaf position. However, $\chi_s$ may differ significantly depending on leaf position (e.g., upper, middle and lower) due to difference in the N status of the leaves. Thus, the resulting effects of enhanced N and $O_3$ on $\chi_s$ are not enough to represent the characteristics of the entire-plant. Furthermore, the present experiment was designed based on OTC chambers. However, OTCs have effects of their own, such as differences in microclimate (e.g., air temperature and humidity), fixed gas flow and limited space, which could over-estimate or under-estimate the effects of $O_3$ on plants (Feng et al., 2010). Note that $\chi_s$ is shown to be influenced by air temperature, partly by affecting the amount of $NH_3$ dissolved in the apoplast, and partly by affecting the leaf tissue $NH_4^+$ generation (or assimilation)-associated physiological processes (Schjoerring et al., 1998). To more accurately assess the effects of N, $O_3$ and/or plant growth stage on $\chi_s$, at least the two following developments are recommended in future work: 1) to employ open-air fumigations ($O_3$-FACE systems, Paoletti et al., 2016), and 2) to investigate vertical profile of $\chi_s$ at plant scale

## 4.6. Conclusions and Implications

In summary, the current study is the first time to investigate the combined effects of $O_3$ exposure and use of urea as N load on $\chi_s$ of forest species (i.e., poplar clone 546). Our results demonstrated that elevated $O_3$ significantly reduced $\chi_s$, in tandem with $A_{\text{sat}}$,
Ci and Chl. Also, urea addition significantly reduced χs, and can mitigate the negative effects of O3 on χs (Fig. 3, Table 1). Our findings filled knowledge gaps in the influence of O3 on χs, and provided valuable and additional information on effect of N addition on χs, which is commonly expected to increase considerably with input of the different types of inorganic N fertilizer applied (Massad et al., 2010). In addition, we demonstrated a significant reduction of χs in the context of O3-accelerated senescence, in contrast to the common view that χs is affected by the plant's development stage and may peak at senescence (Hill et al., 2002; Mattsson and Schjoerring, 2003). Based on these findings, we propose that current parameterizations of χs in chemical transport models should be optimized to partially respond to changes in environmental conditions (e.g., elevated N and/or O3). In addition, Asat, gs and Chl significantly and positively corrected with χs, whereas a significant and negative relationship was observed between Ci and χs (Fig. 5). Understanding these physiological controls of χs is essential for modeling its dynamic behaviour.

566

## Acknowledgments

This study was supported by the National Natural Science Foundation of China (41705130), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC019), the National Key R&D Program of China (2017YFC0210106), the Hundred Talents Program, Chinese Academy of Sciences.

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837 **Figure captions**

838 Figure 1. Effects of ozone (CF, charcoal-filtered ambient air, and E-O₃, elevated O₃) and Nitrogen (N0, no N added, and N50, 50 kg N ha⁻¹ yr⁻¹) on light-saturated photosynthesis (Aₘsat), stomatal conductance (gₘ), intercellular CO₂ concentration (Cᵢ) and chlorophyll (Chl) content of hybrid poplar clone ‘546’ (*Populus deltoides* cv. 55/56 x *P. deltoides* cv. *Imperial*). Data shown are the mean ± standard deviation of three-OTC measurements. The letters on top of the bars are based on the Tukey test across the two measurements, with different letters indicating significantly different from each other at *P*<0.05.

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846 Figure 2. Effects of ozone (CF, charcoal-filtered ambient air, and E-O3, elevated O3) and Nitrogen (N0, no N added, and N50, 50 kg N ha⁻¹ yr⁻¹) on apoplastic NH₄⁺ concentration, tissue NH₄⁺ concentration, apoplastic pH and emission potential (Γs) of hybrid poplar clone ‘546’ (Populus deltoides cv. 55/56 x P. deltoides cv. Imperial). Data shown are the mean ± standard deviation of three-OTC measurements. The letters on top of the bars are based on the Tukey test across the two measurements, with different

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letters indicating significantly different from each other at *P*<0.05.

Figure 3. Effects of ozone (CF, charcoal-filtered ambient air, and E-O₃, elevated O₃) and Nitrogen (N0, no N added, and N50, 50 kg N ha⁻¹ yr⁻¹) on the stomatal compensation point (χs)

Figure 4. Correlation between the stomatal compensation point (χs) and light-saturated photosynthesis (Asat), stomatal conductance (gs), intercellular CO₂ concentration (Ci) and chlorophyll (Chl) content across all ozone and nitrogen treatments. Green and red dots represent charcoal-filtered ambient air (CF)*N0 (no N added), CF*N50 (50 kg N ha⁻¹ yr⁻¹), elevated O₃ (E-O₃)*N0 and E-O₃*N50 treatment in July and August, respectively. While ANCOVA did not show significant differences in the slope of the regression lines for the relationships between the two measurement dates (i.e., July and August), one single line is shown; otherwise, two lines are shown.

Figure 5. Correlations between apoplastic pH and light-saturated photosynthesis (Asat), chlorophyll (Chl) content, and intercellular CO₂ concentration (Ci), and correlation between apoplastic NH₄⁺ concentration and leaf tissue NH₄⁺ concentration across all ozone and nitrogen treatments. Green and red dots represent charcoal-filtered ambient air (CF)*N0 (no N added), CF*N50 (50 kg N ha⁻¹ yr⁻¹), elevated O₃ (E-O₃)*N0 and E-O₃*N50 treatment in July and August, respectively. While ANCOVA did not show significant differences in the slope of the regression lines for the relationships between the two measurement dates (i.e., July and August), one single line is shown; otherwise, two lines are shown.

Figure 6. Actual forest distribution in China (a) (adopting from Li et al. (2017)) and atmospheric NH₃ concentration over deciduous broadleaf forests in July (b) and August (c) modeled using the GEOS-Chem model.
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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

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<caption>Figure 2</caption>
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

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<table><caption>**Table 1.** ANOVA results (*P* values) for the individual effects of interactions of O₃ (CF and E-O₃), N (N0 and N50), and sampling data (July and August) on light-saturated rate of CO₂ assimilation (*A*<sub>sat</sub>), stomatal conductance (*g*<sub>s</sub>), intercellular CO₂ concentration (*Ci*), chlorophyll content (*Chl*), apoplastic pH, apoplastic NH₄⁺, leaf tissue NH₄⁺, potential emission (*r*<sub>s</sub>), and stomatal compensation point (*χ*<sub>s</sub>).</caption><thead><tr><th></th><th>O<sub>3</sub></th><th>N</th><th>Date (D)</th><th>O<sub>3</sub>*N</th><th>O<sub>3</sub>*D</th><th>N*D</th><th>O<sub>3</sub>*N*D</th></tr></thead><tbody><tr><td>A<sub>sat</sub></td><td><strong>&lt;0.001</strong></td><td>0.057</td><td><strong>0.001</strong></td><td>0.977</td><td>0.553</td><td><strong>0.042</strong></td><td>0.063</td></tr><tr><td>g<sub>s</sub></td><td>0.610</td><td>0.579</td><td><strong>0.003</strong></td><td>0.285</td><td><strong>0.048</strong></td><td>0.843</td><td>0.564</td></tr><tr><td>Ci</td><td><strong>0.007</strong></td><td>0.257</td><td><strong>&lt;0.001</strong></td><td>0.192</td><td><strong>0.002</strong></td><td>0.404</td><td>0.295</td></tr><tr><td>Chl</td><td><strong>0.006</strong></td><td>0.612</td><td><strong>0.001</strong></td><td>0.727</td><td>0.527</td><td>0.375</td><td>0.658</td></tr><tr><td>Apoplast pH</td><td><strong>0.015</strong></td><td><strong>0.018</strong></td><td><strong>&lt;0.001</strong></td><td>0.118</td><td>0.240</td><td>0.657</td><td>0.391</td></tr><tr><td>Apoplast NH<sub>4</sub><sup>+</sup></td><td><strong>0.039</strong></td><td><strong>0.017</strong></td><td><strong>0.004</strong></td><td>0.996</td><td>0.431</td><td>0.172</td><td>0.698</td></tr><tr><td>Leaf tissue NH<sub>4</sub><sup>+</sup></td><td><strong>0.041</strong></td><td>0.965</td><td><strong>0.003</strong></td><td>0.265</td><td>0.219</td><td>0.097</td><td>0.210</td></tr><tr><td>r<sub>s</sub></td><td><strong>0.026</strong></td><td>0.067</td><td><strong>&lt;0.001</strong></td><td>0.062</td><td>0.318</td><td>0.777</td><td>0.369</td></tr><tr><td>χ<sub>s</sub></td><td><strong>0.019</strong></td><td><strong>0.034</strong></td><td><strong>&lt;0.001</strong></td><td><strong>0.032</strong></td><td>0.203</td><td>0.322</td><td>0.337</td></tr></tbody></table>

920 CF: charcoal-filtered ambient air; E-O₃: elevated O₃; N0: no N added; N50: 50 kg N
921 ha⁻¹ yr⁻¹; Statistically significant effects (*P*<0.05) are marked in bold.

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