Mesophyll conductance limitation of photosynthesis in

Science of the Total Environment 657 (2019) 136–145

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Mesophyll conductance limitation of photosynthesis in poplar under elevated ozone Yansen Xu a,b,c, Zhaozhong Feng a,b,⁎, Bo Shang a,b, Lulu Dai a,b, Johan Uddling c, Lasse Tarvainen c a b c

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Department of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The photosynthetic response to elevated ozone was investigated in poplar trees. • Ozone exposure reduced leaf mesophyll conductance and carboxylation capacity. • Stomatal conductance was unaffected by elevated ozone. • The overall photosynthetic limitation increased with exposure time. • The main ozone-induced limitation to photosynthesis was reduced mesophyll conductance.

a r t i c l e

i n f o

Article history: Received 2 September 2018 Received in revised form 6 November 2018 Accepted 30 November 2018 Available online xxxx Editor: Yasutomo Hoshika Keywords: Carboxylation Mesophyll conductance Ozone Poplar Photosynthesis Stomatal conductance

a b s t r a c t Finite mesophyll conductance (gm) reduces the rate of CO2 diffusion from the leaf intercellular space to the chloroplast and constitutes a major limitation of photosynthesis in trees. While it is well established that gm is decreased by stressors such as drought and high temperature, few studies have investigated if the phytotoxic air pollutant ozone (O3) affects gm. We quantified the relative importance of three different types of limitations of photosynthesis in poplar trees exposed to elevated O3: decreases in stomatal conductance, gm and biochemical photosynthetic capacity. The O3-induced reductions in light-saturated net photosynthesis were linked to significant declines in gm and biochemical photosynthetic capacity (in particular carboxylation). There was no significant effect of O3 on stomatal conductance. Of the O3-induced limitations on photosynthesis, gm limitation was by far the most important (−16%) while biochemical limitation (−8%) was rather small. Both limitations grew in magnitude over the study period and varied in response to leaf-specific O3 exposure. Our findings suggest that declines in gm may play a key role in limiting photosynthesis of plants exposed to elevated O3, an effect hitherto overlooked. © 2018 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author at: State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. E-mail address: [email protected] (Z. Feng).

https://doi.org/10.1016/j.scitotenv.2018.11.466 0048-9697/© 2018 Elsevier B.V. All rights reserved.

Tropospheric ozone (O3) is a detrimental phototoxic air pollutant that enters leaves through stomata and causes physiological oxidative stress and growth reductions in plants, including both crops and trees

Y. Xu et al. / Science of the Total Environment 657 (2019) 136–145

(Feng et al., 2015; Li et al., 2017; Paoletti and Manning, 2007; Wittig et al., 2009). Ozone-induced reductions of net photosynthesis (An) appear to play a key role in driving losses in tree growth and forest productivity (Yue et al., 2017). However, there is no consensus if this decrease in An is primarily caused by negative effects of O3 on stomatal conductance (gs), biochemical photosynthetic capacity, mesophyll conductance for CO2 diffusion from sub-stomatal cavities to chloroplasts (gm), or a combination of these effects (Calatayud et al., 2007; Feng et al., 2016; Gao et al., 2016). Stomatal closure responses limit photosynthesis by decreasing the diffusion of CO2 from the atmosphere surrounding the leaf to the substomatal intercellular cavities. In a global meta-analysis of experiments with trees, ambient O3 concentrations (42 ppb) decreased gs by 16% on average compared to trees grown in charcoal-filtered clean air (Wittig et al., 2007). Another meta-analysis on woody species in China reported that elevated O3 (102 ppb) decreased gs by 29% across experiments (Li et al., 2017). However, although lower gs generally decreases An in C3 plants, it does not necessarily result in increased relative stomatal limitation of An, which also depends on the magnitude of possible cooccurring changes in An. In most experiments with trees, O3 caused a greater decrease in An than in gs, leading to increased CO2 concentration in the leaf intercellular spaces (Ci), and thus decreased gas-phase limitations of An (Li et al., 2017; Wittig et al., 2007). This decoupling of photosynthetic and stomatal regulation under elevated O3—which has been attributed to stomatal sluggishness (i.e. loss of stomatal sensitivity and control over transpiration; Hoshika et al., 2014)—indicates that O3induced reductions in An are not primarily linked to stomatal limitations (Paoletti and Grulke, 2005). A significant part of the effect of O3 on An is likely to be caused by impairment of photosynthetic biochemistry. Elevated O3 often decreases the maximum rates of Rubisco carboxylation (Vcmax; Ainsworth et al., 2012; Gao et al., 2016; Li et al., 2017; Wittig et al., 2009). In fact, this effect has often been suggested to play the main role in decreasing An under elevated O3 (Wallin et al., 1992; Reichenauer et al., 1997; Noormets et al., 2001; Watanabe et al., 2013). The other main biochemical trait determining photosynthetic capacity is the maximum rate of ribulose 1,5‑bisphosphate (RuBP) regeneration in the Calvin cycle (Jmax), which is related to the maximum rate of photosynthetic electron transport. There are conflicting results on how elevated O3 affects Jmax, with some studies not detecting any significant effects (Farage and Long, 1999; Morgan et al., 2004) and other studies reporting negative effects (Niu et al., 2014; Zheng et al., 2002). Chlorophyll fluorescence measurements, however, have shown consistent but usually rather small negative effects of O3 on the maximum potential PSII quantum yield (Chutteang et al., 2016; Gao et al., 2016). Previous research thus indicates that O3 impairs photosynthetic biochemistry and that effects on Rubisco carboxylation seem more important than those on electron transport and RuBP regeneration. Calculations of the biochemical capacities Vcmax and Jmax have usually been based on Ci, and thus on the assumption of effectively infinite conductance of CO2 diffusion from the intercellular space to chloroplasts (Farquhar et al., 1980), i.e. infinite gm. However, it has been shown that gm is finite, which leads to a substantial CO2 drawdown from Ci to the CO2 concentration at the chloroplast (Cc; Niinemets et al., 2009), thereby constituting a major limitation of photosynthesis in most tree species (Peguero-Pina et al., 2017). A large body of evidence indicates that ignoring the finite gm leads to underestimations of the actual Vcmax and, to a lesser extent, Jmax (Flexas et al., 2008; Singsaas et al., 2004; Warren et al., 2007). Accordingly, inclusion of finite gm has improved the correlation between Vcmax estimates and in vitro Rubisco measurements (Singsaas et al., 2004). Numerous studies have examined how gm responds to different environmental variables, e.g. CO2, drought, light availability and temperature (Flexas et al., 2008; Caemmerer and Evans, 2015). It was suggested that O3 might decrease gm through its effects on mesophyll structure, e.g. cell wall thickening or altered chloroplast size, shape and location (Paoletti et al., 2009). If

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so, it is possible that a significant part of the O3-induced reductions in Vcmax and Jmax described in the preceding paragraph (which were based on Ci rather than Cc) was actually caused by decreased gm rather than decreased biochemical capacity. So far, few studies have investigated the effect of O3 on gm and those conducted have yielded inconclusive results. Elevated O3 caused a decline in gm in the O3 sensitive species soybean, snap bean, silver birch, oak and Siebold's beech (Eichelmann et al., 2004; Velikova et al., 2005; Flowers et al., 2007; Sun et al., 2014; Watanabe et al., 2018) while gm was unaffected by O3 in European beech (Warren et al., 2007). The earlier studies on O3 sensitive species did not quantify the degree to which the decrease in gm contributed to the decrease in An by elevated O3. Neither did they determine how the gm limitation compared to biochemical limitations (i.e. values of Vcmax and Jmax based on Cc) in explaining the decrease in An under elevated O3. The overall aim of this study is to explore the relative contributions of gs, gm and biochemical limitations in explaining the reduction in An under elevated O3. An O3 sensitive clone of poplar was exposed to charcoal-filtered ambient air (CF) or elevated O3 (ambient concentration + 40 ppb) and measurements of leaf gas exchange and chlorophyll fluorescence were conducted at four times during the growing season to address the following two research questions: (1) Does elevated O3 significantly decrease gm in the O3 sensitive poplar clone? (2) Is gm limitation a major contributor to O3-induced reductions in An, equally or more important than the contributions by gs and biochemical photosynthetic capacity? 2. Material and methods 2.1. Experimental site and plant materials The experimental site is located in Yanqing (40°29′N, 115°60′E), northwest of Beijing, China. It has a warm temperate and semi-humid continental climate, typical for its region. The annual mean temperature for the period 2006–2016 in Yanqing was 9.9 °C and the warmest month was July with a mean temperature of 24.5 °C. Average annual precipitation was 467 mm, with about 44% falling between June and September. Rooted cuttings of the O3-sensitive hybrid poplar clone ‘546’ (Populous deltoides cv. 55/56 × P. deltoides cv. Imperial) were cultivated in six open-top-chambers (OTCs, octagonal base, 12.5 m2 of growth space and 3.0 m of height, covered with toughened glass) on April 6, 2017. They were individually transplanted into 20-liter circular plastic pots filled with a mixture consisting of equal volumes of native brown sandy loam soil and planting soil consisting of organic matter and sand on May 7, 2017. The brown sandy loam soil was excavated at 0–10 cm depth from a nearby farm, sieved out by a 3 mm pore mesh and then carefully mixed for homogeneity. Plants selected for the study had similar initial heights and basal stem diameters. 2.2. Ozone exposure Ozone fumigation lasted from June 10 until September 22 in 2017. Two O3 treatments were applied: CF and non-filtered ambient air with a targeted O3 addition of 40 ppb during 8:00 to 18:00 h (E-O3). Each treatment had three replicate OTCs and each OTC had 32 plants that were well watered during the experiment. Ozone was generated from pure oxygen using an O3 generator (HY003, Chuangcheng Co., Jinan, China) and mixed with ambient air by a fan (1.1 KW, 1080 Pa, 19 m3 min−1, CZR, Fengda, China) to achieve the target O3 concentration at the top of the canopy. O3 concentrations were continuously monitored in all OTCs. Air was taken from the sampling point at approximately 10 cm above the canopy to an UV absorption O3 analyzer (Model 49i, Thermo Scientific, USA) via a teflon solenoid valve switch system. The monitors were calibrated by a 49i-PS calibrator (Thermo Scientific, USA) before the experiment and once a month during the experiment. Fumigation was running when there was no rain, fog,

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Table 1 Different measures of O3 exposure: M10 (the mean of the hourly O3 concentration from 08:00 to 18:00) and AOT40 (accumulated O3 exposure over an hourly threshold concentration of 40 ppb) for the entire experiment as well as for leaves measured at each sampling occasion. The leaf age at each sampling occasion is also shown. CF and E-O3: plants grown in charcoalfiltered ambient air or elevated O3, respectively. S1–S4: sampling campaigns. Values shown are means ± SE. Leaf age (days)

S1 S2 S3 S4

26 26 26 54

CF

E-O3

M10 (ppb)

AOT40 (ppm·h)

Leaf-specific AOT40 (ppm·h)

M10 (ppb)

AOT40 (ppm·h)

Leaf-specific AOT40 (ppm·h)

32.8 ± 1.2 29.1 ± 1.0 27.4 ± 0.8 25.1 ± 0.5

1.3 ± 0.2 1.8 ± 0.2 2.1 + 0.2 2.3 ± 0.2

1.3 ± 0.2 0.9 ± 0.1 0.6 + 0.1 0.8 ± 0.1

99.7 ± 1.3 92.4 ± 0.8 89.5 ± 1.7 82.7 ± 1.8

13.6 ± 0.3 21.2 ± 0.3 27.7 ± 0.9 36.6 ± 1.5

13.6 ± 0.3 11.9 ± 0.1 9.9 ± 0.8 18.8 ± 1.4

mist, or dew, according to the protocols of free air O3 concentration enhancement systems (Feng et al., 2016). During 8:00–18:00 h in the experimental period, the mean O3 concentrations were 24 and 81 ppb, the hourly peak O3 concentrations were 111 and 194 ppb, and values of AOT40 (accumulated O3 exposure over an hourly threshold concentration of 40 ppb) were 2.4 and 41.6 ppm h in CF and E-O3, respectively. The daily average air temperatures inside and outside the OTCs were 22.4 and 20.5 °C during ozone fumigation hours, respectively. 2.3. Gas exchange and chlorophyll fluorescence measurements Leaf gas exchange and chlorophyll fluorescence were measured simultaneously using an open gas exchange system Li-6400 (LI-COR Inc., Lincoln, NE) with an integrated fluorescence chamber head (Li-640040) from 9:00 to 15:00 h. For each OTC, fully expanded mid-canopy leaves from two randomly selected trees were measured for their photosynthetic CO2 responses with one Li-6400 at four sampling campaigns (S1– S4): June 29 to July 5, S1; July 18 to July 25, S2; August 5 to August 10, S3; September 1 to September 7, S4. Table 1 shows the mean of the hourly O3 concentration from 08:00 to 18:00 (M10) and AOT40 of leaves measured at each sampling time (S1–S4) and in both CF and E-O3 treatments. During the A-Ci measurements, the photosynthetic photon flux density (PPFD) was kept at 1200 μmol m−2 s−1, the block temperature was set at prevailing environmental conditions (27–34 °C, 25–34 °C, 24–33 °C and 20–30 °C at sampling times S1–S4, respectively) and relative humidity was kept at 50–60%. The A-Ci measurements were started when gs had stabilized to these conditions (usually in 30 min after clamping onto the leaf). The ambient CO2 concentration (Ca) was adjusted in a series of 400, 300, 250, 200, 150, 100, 50, 400, 500, 700, 1000, 1300, 1600 μmol mol−1. During each step of CO2 change, the minimum and the maximum waiting times were 4 and 6 min, respectively. The measurement at ambient Ca (400 μmol mol−1) was used for determinations of light-saturated net photosynthesis (Asat, μmol m−2 s−1), stomatal conductance to CO2 (gs, mol CO2 m−2 s−1), Ci (μmol mol−1) and chlorophyll fluorescence parameters. The quenching of photochemical efficiency of PSII (qP), the actual photochemical efficiency of PSII in the saturated light (Fv′/Fm′) and the actual quantum efficiency of PSII (ΦPSII) were determined from measurements of steady-state fluorescence (Fs) at 1200 μmol m−2 s−1 and maximum fluorescence (Fm′) during a light-saturating pulse of ca. 8000 μmol m−2s−1 by the multiphase flash protocol: 0 0 ΦPSII ¼ F m − F s =F m

ð1Þ

The electron transport rate (Jflu) at 1200 μmol m−2 s−1 was estimated as follows: J flu ¼ ΦPSII PPFD α β

ð2Þ

where α is the leaf absorbance and β is the distribution of absorbed energy between photosystem I and II. The product αβ was determined from the slope of the relationship between An and (PPFD·ΦPSII/4), which was determined from light response measurements under nonphotorespiratory conditions (Yin and Struik, 2009) during the second sampling occasion. The value 4 originates from the assumption of 4

electrons being required for each Rubisco carboxylation in the biochemical photosynthesis model of Farquhar et al. (1980). PPFD was adjusted in a series of 200, 150, 100, 75 and 30 μmol m−2 s−1, while keeping Ca at 400 μmol mol−1 and O2 at 2% at ambient temperature. During each step of PPFD change, the minimum and the maximum waiting times were 3 and 5 min, respectively. For the measurements at 2% O2 and 98% N2, mixed gas was humidified and supplied to the Li-6400 where CO2 was blended with the mixed gas. The αβ values were significantly related to observed SPAD values (R2 = 0.43, P b 0.001; Fig. S1), which provide a relative measure of leaf chlorophyll content and were obtained using a SPAD-502 chlorophyll meter (Konica Minolta, Osaka, Japan). This relationship was used to estimate αβ based on SPAD measurements made during the other sampling campaigns, when the αβ values were not directly determined (Table S1). The dark respiration rate (Rdark), the maximum quantum efficiency of PSII (Fv/Fm) and the non-photochemical quenching (qN) were measured on the leaves used for A-Ci curves from 21:00 to 23:00 h on the same day using the Li-6400-40s. The CO2 concentration in the leaf chamber was set to 400 μmol mol−1 and air humidity and temperature were kept at ambient levels. Leaves measured for gas exchange were also sampled for determination of leaf mass per area (LMA) based on five leaf discs of known area (1.13 cm2) dried at 80 °C to constant mass.

Table 2 ANOVA results (P values) for main effects and interactions of ozone and sampling time on leaf traits. Significant effects (P b 0.05) are marked in bold. Leaf traits

Time

Ozone

Ozone × time

LMA Asat gs gm Ci Cc gtot Rdark Vcmax Jmax Jmax/Vcmax Vcmax (Ci) Jmax (Ci) Jmax/Vcmax (Ci) Fv′/Fm′ ΦPSII Jflu Fv/Fm qP qN

b0.001 b0.001 b0.001 b0.001 0.082 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001

0.040 0.018 0.569 0.018 0.119 0.307 0.030 0.541 0.006 0.940 0.001 0.014 0.896 b0.001 0.032 0.022 0.007 b0.001 0.105 0.090

0.005 0.685 0.909 0.426 0.609 0.902 0.806 0.245 0.866 0.642 0.027 0.845 0.584 0.336 0.562 0.297 0.529 0.883 0.442 0.416

LMA: leaf mass per area, Asat: light-saturated net photosynthesis, gs: stomatal conductance to CO2, gm: mesophyll conductance for CO2, Ci: CO2 concentration in the leaf intercellular spaces, Cc: CO2 concentration at the chloroplast, gtot: total conductance to CO2, Rdark: dark respiration rate, Vcmax: the maximum rates of Rubisco carboxylation, Jmax: the maximum rate of ribulose 1,5‑bisphosphate regeneration, Vcmax (Ci): the maximum rate of Rubisco carboxylation calculated based on Ci, Jmax (Ci): the maximum rate of ribulose 1,5‑bisphosphate regeneration calculated based on Ci, Fv′/Fm′: the actual photochemical efficiency of PSII in the saturated light, ΦPSII: the actual quantum yield of PSII, Jflu: the electron transport rate, Fv/Fm: the maximum quantum yield of PSII, qP: the quenching of photochemical efficiency of PSII, qN: the quenching of non-photochemical efficiency. The values of Vcmax and Jmax were rescaled to 25 °C before the statistical analyses.


2.4. Estimation of mesophyll conductance and biochemical parameters By combining gas exchange and chlorophyll fluorescence measurements, gm was estimated using the variable J method of Harley et al. (1992), as follows: gm ¼

AN Γ ð J flu þ 8ðAN þ Rd ÞÞ Ci − J flu −4ðAN þ Rd Þ

ð3Þ

Values of An and Ci were taken from the gas-exchange measurements. Following Théroux-Rancourt et al. (2014) who studied four hybrid poplar clones, we assumed the value of Г⁎ (i.e. the CO2

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compensation point in the absence of mitochondrial respiration) to be 38 μmol mol−1 at 25 °C. This assumption was originally based on in vitro determinations of the specificity factor (Sc/o) from other woody deciduous species (Vitis vinifera and Quercus robur) (Balaguer et al., 1996; Bota et al., 2002). The Rd term represents the rate of mitochondrial respiration in the light and was taken as half of the measured Rdark (Way and Yamori, 2014). Temperature response functions for Г⁎ and Rd were taken from Bernacchi et al. (2002) and Bernacchi et al. (2001), respectively. Using estimates of gm, A-Ci curves could be converted into A-Cc curves. In addition, we attempted to estimate gm from A-Ci curves (Ethier and Livingston, 2004) to compare with the gm estimates produced by the variable J method. However, the A-Ci curvebased gm analyses yielded unrealistically high results compared to

Fig. 1. Effects of ozone and sampling time on (a) leaf mass per area (LMA), (b) light-saturated net photosynthesis (Asat), (c) dark respiration rate (Rdark), (d) total conductance to CO2 (gtot), (e) stomatal conductance to CO2 (gs), (f) mesophyll conductance for CO2 (gm), (g) CO2 concentration in the leaf intercellular spaces (Ci) and (h) CO2 concentration at the chloroplast (Cc). CF and E-O3: plants grown in charcoal-filtered ambient air or elevated O3, respectively. S1–S4: first through fourth sampling campaign. Different letters indicate significant differences between bars (mean ± SE, Tukey HSD, P b 0.05, n = 3).

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previously reported values. Therefore, we only present the data obtained by the variable J method here. Values of Vcmax and the potential electron transport rate (J) were calculated from both A-Cc and A-Ci curves. Jmax was estimated from J and PPFD, as follows: θ J 2 −ðαPPFD þ J max ÞJ þ αPPFDJ max ¼ 0 where Jmax is the potential maximum rate of electron transport. The curvature of the light response curve (θ) and the quantum yield of electron transport (α) are set to 0.9 and 0.3, respectively. For A-Cc curves, values of the Rubisco Michaelis-Menten constants for carboxylation (KC) and oxygenation (KO) were obtained from Bernacchi et al. (2002). For A-Ci curves, these parameters were taken from Bernacchi et al. (2001). The values of Vcmax and Jmax reported in this study were re-scaled to 25 °C using temperature response functions from Bernacchi et al. (2001, 2003). 2.5. Analysis of quantitative limitations of light-saturated photosynthesis rate (Asat)

where d stands for the difference in values between CF and E-O3 and CF values are used in the denominators. 2.6. Statistical analyses The chambers were considered as the statistical units and data from multiple trees in a chamber were averaged prior to statistical analyses. Before the analyses, the Shapiro-Wilk's and Levene's tests were conducted to check all data for normal distribution and homogeneity of variances, respectively. Statistical tests were made on log-transformed data to test for multiplicative rather than additive effects. Repeatedmeasures one-way ANOVA with the Tukey HSD post hoc test was used to test for effects of O3, sampling time and their interaction. The sphericity assumption of covariance matrix was tested using Mauchly's test. All statistical tests were done in R statistical software (version 3.3.3, http://www.r-project.org/), using the “ezANOVA” function in the “ez” package. Linear regressions were fitted with the ‘lm’ function. The slopes and intercepts were tested using ANCOVA. Effects were considered significant at P b 0.05. 3. Results

To separate the relative limitations of Asat resulting from gs (ls), gm (lm), and biochemical photosynthetic capacity (lb), the quantitative limitation analysis of Grassi and Magnani (2005) was used. The relative photosynthesis limitations by the different components; ls, lm and lb (ls + lm + lb = 1) were determined as follows: ls ¼

g tot =g s ∂AN=∂Cc g tot þ ∂AN=∂Cc

ð4Þ

lm ¼

g tot =g m ∂AN =∂C c g tot þ ∂AN =∂C c

ð5Þ

lb ¼

g tot g tot þ ∂AN =∂C c

ð6Þ

where gtot is the total conductance to CO2 from the leaf surface (i.e. inside the leaf boundary layer) to chloroplast (1/gtot = 1/gs + 1/gm). The ∂AN/∂Cc term was estimated as the slope of A-Cc curves over a Cc range of 50–100 μmol mol−1. The contributions of different types of O3 effects on the reduction of Asat—i.e., effects on stomatal conductance (SL), mesophyll conductance (MCL) and biochemical photosynthetic capacity (BL)—were quantified as follows: dA dg dg dV c max ¼ SL þ MC L þ BL ¼ ls s þ lm m þ lb A gs gm V c max

ð7Þ

3.1. Leaf mass per area and gas exchange parameters Overall, LMA increased significantly during the growing season with most of the change occurring from S1 to S2 (Table 2, Fig. 1a). In addition, a decrease in LMA was observed from S2 to S4 for plants grown in E-O3. Accordingly, there was a significant interactive effect of O3 and sampling time on LMA (Table 2). Both CF and E-O3 plants exhibited significant declines in Asat, Rdark, gtot, gs gm, and Cc over the four sampling times, while no change over time was observed in Ci (Fig. 1, Table 2). E-O3 significantly reduced Asat, gm, gtot and Cc (Table 2) with the effects on the gas exchange parameters being most prominent in the last sampling (Fig. 1b, f, g, h). Notably, at this time the gm and gtot were reduced by 52% and 40% in the E-O3 plants compared to the CF plants, respectively (Fig. 1f, d). Averaged over the whole experiment (i.e. all sampling occasions and both treatments), Ci was 145 μmol mol−1 higher than Cc (Fig. 1g, h). Both Ci and Cc increased at later sampling occasions and were unaffected by elevated O3 (Table 2). There were no significant effects of O3 on gs and Rdark (Table 2, Fig. 1e, c). Across the sampling times and treatments, Asat was significantly correlated with gs (R2 = 0.60, P b 0.001; Fig. S2a), gm (R2 = 0.80, P b 0.001; Fig. S2b) and gtot (R2 = 0.91, P b 0.001; Fig. S2c). Furthermore, the values of gs and gm were also significantly linearly related (R2 = 0.28, P = 0.0043; data not shown). Chlorophylls were assayed at the second and fourth sampling times. There was a

Fig. 2. Mesophyll conductance (gm) in relation to (a) chlorophyll content and (b) leaf per mass (LMA). CF and E-O3: plants grown in charcoal-filtered ambient air or elevated O3, respectively. S2 and S4: the second and fourth sampling campaign, respectively. The responses of gm to chlorophyll content (regression slopes) did not differ significantly between the CF and E-O3 treatments (ANCOVA; P = 0.86) while main effects of both chlorophyll content (P = 0.0050) and O3 treatment (P = 0.0026) were highly significant.


141

Jmax estimates followed a similar pattern (slope = 1.45, R2 = 0.96, P b 0.001; data not shown). Since using Cc-based rather than Ci-based estimates affected Vcmax more than Jmax, the relationship between these two parameters was also affected (Fig. S3b, P b 0.001). During the experiment, Vcmax declined progressively in both treatments (Fig. 3a). The Jmax decreased significantly, by 40%, between S1 and S2 but there was no further change after S2 (Fig. 3b, Table 2). The ratio of Jmax to Vcmax increased significantly during the experiment in both treatments (Fig. 3c, Table 2). Moreover, E-O3 caused a significant increase in the Jmax:Vcmax ratio (Table 2) compared to the CF treatment. Pooling all the data, Vcmax was tightly correlated with Asat (R2 = 0.95, P b 0.001, Fig. S4a). A similar but somewhat weaker relationship was found for Asat and Jmax (R2 = 0.81, P b 0.001, Fig. S4b). The chlorophyll fluorescence analyses indicated that Fv′/Fm′, ΦPSII, Jflu, and Fv/Fm decreased significantly during the experiment (Fig. 4a– d) and were lower in the E-O3 treatment (Table 2). qP also significantly declined during the experiment while qN increased (Fig. 4e, f, Table 2). Neither quenching parameter responded to E-O3 (Fig. 4e, f, Table 2). 3.3. Limitation analysis The relative limitations of gs (ls), gm (lm) and biochemical photosynthetic capacity (lb) on photosynthesis are shown in Fig. 5a, b. In the CF treatment, the lm and lb accounted for c. 44% and 37% of the total limitation, respectively, while ls (19%) was considerably less important. In the E-O3 treatment, lm (49%) was the most important relative limitation of photosynthesis, followed by lb (36%) and ls (15%) (Fig. 5b). Notably, lb increased over time and was equal to lm during the last sampling campaign in both treatments. Across all sampling occasions, the decline in Asat of poplar trees grown under E-O3 was caused primarily by decreased gm (−16%), to a considerably smaller extent by decreased biochemical photosynthetic capacity (−8%), and was largely unrelated to gs (−1%; Fig. 6). The effect of decreased gm on Asat (MCL) grew stronger over time (from −10% at S1 to −24% at S4). Similarly, the effect of biochemical limitation on Asat (BL) increased from −3% at S1 to −15% at S4. The effect of O3 on the photosynthetic limitation by gs (SL) did not vary among the sampling campaigns. 4. Discussion

Fig. 3. Effects of ozone and sampling time on (a) the maximum rates of Rubisco carboxylation (Vcmax), (b) the maximum rate of ribulose 1,5‑bisphosphate regeneration (Jmax) and (c) Jmax/Vcmax ratio. CF and E-O3: plants grown in charcoal-filtered ambient air or elevated O3, respectively. S1–S4: first through fourth sampling campaign. All estimates are based on Cc. Different letters indicate significant differences between bars (mean ± SE, Tukey HSD, P b 0.05, n = 3). The values of Vcmax and Jmax have been rescaled to 25 °C.

positive and significant linear relationship between leaf chlorophyll content and gm (Fig. 2a, P = 0.0027). The ANCOVA showed that the slope of the chlorophyll–gm relationship was highly significant (P = 0.0050) and did not differ between CF and E-O3 (P = 0.86), while the two treatments did have different elevations of the relationships (P = 0.0026). No relationship between gm and LMA was observed (P = 0.95; Fig. 2b). 3.2. Biochemical capacity and chlorophyll fluorescence parameters The Vcmax and Jmax values estimated from Cc were 68% and 16% higher, respectively, than corresponding Ci-based estimates, averaged across O3 treatments and sampling periods (compare Fig. 3a, b and supplementary Fig. S3c, a). The Vcmax estimates calculated based on Ci and Cc were linearly related with a slope of 1.94 (R2 = 0.93, P b 0.001; data not shown). The

Previous studies have attributed the negative effects of O3 on photosynthesis to decreased gs (e.g. Kanagendran et al., 2018; Warren et al., 2007) or impairment of photosynthetic biochemistry (e.g. Noormets et al., 2001; Reichenauer et al., 1997; Watanabe et al., 2013). However, few studies have explored the possible role played by O3-induced decline in gm. Combining measurements of leaf gas exchange and chlorophyll fluorescence, we were able to estimate the CO2 concentration at the chloroplast and thereby separate and quantify the influences of gm, biochemical photosynthetic capacity and gs on photosynthesis. Our results demonstrated that the decline in Asat of poplar trees grown under elevated O3 was caused primarily by decreased gm (−16%), to a considerably smaller extent by decreased biochemical photosynthetic capacity (−8%), and not significantly at all by gs (−1%). Possible interpretations and implications of these findings are discussed below. Light-saturated photosynthesis and gm decreased with both seasonal progression and O3 exposure (Fig. 1b and f). The observation that Asat declined from S1 to S4 in both treatments agrees with previous findings suggesting that the seasonal pattern in photosynthetic capacity may be related to changes in day length (Bauerle et al., 2012). Ozoneinduced effects were largest at the last sampling occasion, when elevated O3 decreased Asat, gm and Vcmax by 36%, 52% and 35%, respectively. Previous studies on snap bean, birch and soybean have reported that sensitive clones exhibited O3-induced declines in both gm and biochemical photosynthetic capacity (i.e. Cc-based Vcmax) while tolerant clones lacked effects on both traits (Eichelmann et al., 2004; Flowers et al.,

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Fig. 4. Effects of ozone and sampling time on (a) the actual photochemical efficiency of PSII in the saturated light (Fv′/Fm′), (b) the actual quantum yield of photosystem II (ΦPSII), (c) the electron transport rate (Jflu), (d) the maximum quantum yield of PSII (Fv/Fm), (e) the quenching of photochemical efficiency of PSII (qP) and (f) the quenching of non-photochemical efficiency (qN). CF and E-O3: plants grown in charcoal-filtered ambient air or elevated O3, respectively. S1–S4: first through fourth sampling campaign. Different letters indicate significant differences between bars (mean ± SE, Tukey HSD, P b 0.05, n = 3).

2007; Sun et al., 2014). In contrast, neither gm nor Vcmax were affected in a study 60-year-old Fagus sylvatica where O3-induced negative effects on Asat were instead caused by decreased gs (Warren et al., 2007). It thus appears that O3-induced inhibition in gm coincides with impairment of photosynthetic biochemistry, such that both appear at a certain stage where detoxification capacity is exceeded and damage starts to accumulate in mesophyll cells. Mesophyll conductance is influenced by three main components: conductance through the intercellular air space, through the cell wall and through the liquid phase inside cells (Flexas et al., 2008; Niinemets et al., 2009). Possible mechanisms by which elevated O3 could decrease gm include cell wall thickening or altered chloroplast amount, size, shape and location (Paoletti et al., 2009). In our study, we observed a strong and positive linear relationship between leaf chlorophyll content and gm (R2 = 0.57, P = 0.0027; Fig. 2a) and that elevated O3 significantly decreased chlorophyll content at the fourth sampling times (data not shown). We did not measure chloroplast structure or location. Previous research has shown that elevated O3 decreases the size and surface area of individual chloroplasts (Fares et al., 2006; Kivimaenpaa et al., 2005; Rinnan and Holopainen, 2004) but little is known regarding how O3 affects the location of chloroplasts within mesophyll cells. A study with Norway spruce showed that chloroplasts were more evenly distributed and less clustered in elevated O3 but it did not investigate location in relation to the cell membrane

(Kivimaenpaa et al., 2014). The chloroplast surface area exposed to the cell membrane is a key determinant of CO2 conductance through the liquid phase inside cells (Li et al., 2009; Wang et al., 2017; Xiong et al., 2015). It is likely that the O3-induced reduction in total chlorophyll content observed in the present study was associated with decreased chloroplast size and surface area, which in turn is known to be linked to decreased liquid-phase conductance for CO2 (Bondada and Syvertsen, 2003; Li et al., 2009). However, since we measured only total chlorophyll content, the direct influences of possible changes in chloroplast size, shape and location need to be further explored in future studies. In our study, gm variation among leaves was not significantly related to LMA (P = 0.95; Fig. 2b). Increased cell wall thickness, which is typically linked to higher LMA (Tomas et al., 2013), is thus not a likely cause of decreased gm by elevated O3 in our study. We did not investigate how elevated O3 affected the levels of leaf carbonic anhydrase (CA) or aquaporins, which may also influence gm (Flexas et al., 2012). CA plays a key role in the interconversion between CO2 and HCO3− (Evans et al., 2009) and acts to enhance the apparent CO2 diffusion gradient in mesophyll cells (Terashima et al., 2011). Ozone can decrease pH in both the apoplast and cytoplast, which may decrease CA activity and, thus, gm (Xu et al., 2018). Aquaporins facilitate CO2 diffusion through cytomembranes (Flexas et al., 2006) but it is unclear how they are affected by O3. Neither did we specifically assess the possible effects of elevated


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Fig. 5. Relative stomatal, mesophyll conductance, and biochemical limitations of light-saturated net photosynthesis (Asat) in (a) CF and (b) E-O3. The three limitations together add up to 100% at each sampling occasion. CF and E-O3: plants grown in charcoal-filtered ambient air or elevated O3, respectively. S1–S4: first through fourth sampling campaign. Different letters indicate significant differences between bars (mean ± SE, Tukey HSD, P b 0.05, n = 3).

O3 on CO2 conductance through the intercellular air space. It is possible that O3-induced patchiness in gs across the leaf surface increased the pathlength for CO2 molecules within leaf gas phase. Patchiness in both gs and photosynthesis is common in plants (Terashima et al., 1988) and O3-induced leaf damage is often patchy (Paoletti and Grulke, 2005). However, the effect of this on gm is hard to assess without data on how the fine-scale heterogeneity of both gs and photosynthetic biochemical capacities across the leaf surface changed upon O3 exposure. Leaves of our poplar trees did not exhibit O3-induced necrotic stippling, indicating that patchiness on the scale visible to the human eye was not substantial. Elevated O3 significantly impaired photosynthetic biochemistry as manifested by reductions in Vcmax and most parameters determined by the chlorophyll fluorescence measurements (Figs. 2a and 3). The O3-induced decreases in Jflu, ΦPSII, Fv′/Fm′ and qP likely reflect decreased energy demand of a damaged Calvin cycle, since gas exchange data showed that Vcmax was decreased while Jmax was not significantly so (Fig. 3a–b). This imbalance between light-driven electron transport and Rubisco capacity—manifested by the increased Jmax to Vcmax ratio (Fig. 3c)—supports the notion that negative effects of elevated O3 on photosynthetic biochemistry are primarily caused by decreased Rubisco carboxylation (Dann and Pell, 1989; Farage and Long, 1999; Noormets et al., 2001; Morgan et al., 2004). Moreover, elevated O3 also impaired

the quantum yield of PSII, as shown by the small but significant decrease (3% to 5%) in dark-adapted Fv/Fm (Fig. 4d). The finding that gm was the main contributor to O3-induced reductions in Asat (Fig. 6) has large potential implications. Most importantly, it questions the common view that the main effect of O3 on photosynthesis is caused by declines in Rubisco quantity and/or activity (e.g., Dann and Pell, 1989; Wallin et al., 1992; Noormets et al., 2001; Watanabe et al., 2013). That view is to a large extent (but not entirely, see Dann and Pell, 1989) based on gas exchange observations showing that O3 decreased photosynthetic carboxylation (its efficiency or maximum rate, Vcmax) more than it inhibited photosynthetic light reactions (quantum yield or Jmax). However, in these previous estimates of carboxylation efficiency, Vcmax estimates were based on Ci rather than Cc. This implies that O3-induced negative effects on Vcmax could reflect changes in both gm and carboxylation capacity. However, since gm was for a long time assumed to be high—such that Cc approximately equaled Ci (Laisk, 1977; Farquhar et al., 1980)—the interpretation has been that the O3 effect was occurring at the Rubisco level. Our study shows that O3-induced decreases in gm can actually be more important than impaired photosynthetic biochemistry for limiting photosynthesis of poplar under elevated O3. 5. Conclusion To the best of our knowledge this is the first study that quantifies and compares the relative contributions of gm, biochemical capacity and gs in limiting photosynthesis of plants under elevated O3. We found that elevated O3 decreased both Vcmax and gm while it did not affect gs in the O3 sensitive poplar clone used. Decreased gm was the most important contributor to O3-induced declines in photosynthesis (on average −16%), while biochemical limitation (on average −8%) was rather small. Both limitations grew in magnitude over the study period and varied in response to leaf-specific O3 exposure. Our findings highlight that gm may play a key role in limiting photosynthesis of plants exposed to elevated O3, a hitherto overlooked effect that deserves further investigation in future experiments. Acknowledgements

Fig. 6. Contributions of stomatal conductance limitation (SL), mesophyll conductance limitation (MCL) and biochemical limitation (BL) to decrease Asat in elevated O3 compared to CF treatment. S1–S4: first through fourth sampling campaign.

This study was funded by the National Natural Science Foundation of China (No. 41771034 & 31500396), Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC019) and the Hundred Talents Program, Chinese Academy of Sciences. Support was also provided by the China-Sweden research project ‘Photochemical smog in China: formation, transformation, impact and abatement strategies’ as well as by (for the authors LT and JU) the Swedish strategic research area BECC

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(Biodiversity and Ecosystem Services in a Changing Climate; http:// www.becc.lu.se/). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.11.466.

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