Elevated ozone reduced leaf nitrogen allocation to

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Dec 3, 2018 - Bo Shang a,b, Yansen Xu a,b, Lulu Dai a,b, Xiangyang Yuan a,b, Zhaozhong Feng a,b,c,⁎ a State Key Laboratory of Urban and Regional ...
Science of the Total Environment 657 (2019) 169–178

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

Elevated ozone reduced leaf nitrogen allocation to photosynthesis in poplar Bo Shang a,b, Yansen Xu a,b, Lulu Dai a,b, Xiangyang Yuan a,b, Zhaozhong Feng a,b,c,⁎ a

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District, Beijing 100085, China College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Technology, Nanjing University of Information Science & Technology, Nanjing 210044, China

b c

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

• Ozone exposure-response relationships for leaf N concentration were established. • E-O3 increased Nmass of leaf, but reduced Narea. • E-O3 reduced leaf N allocation to photosynthesis (productivity). • E-O3 increased leaf N allocation to cell walls (defense).

a r t i c l e

i n f o

Article history: Received 22 September 2018 Received in revised form 30 November 2018 Accepted 30 November 2018 Available online 03 December 2018 Editor: Yasutomo Hoshika Keywords: Cell walls Leaf nitrogen concentration Leaf nitrogen allocation Ozone Photosynthesis PNUE

a b s t r a c t We investigated the effects of elevated ozone (O3) concentration on leaf nitrogen (N), a key determinant of plant photosynthesis, with two clones of poplar grown in open-top chambers. We focus on the difference between mass-based leaf N concentration (Nmass) and area-based one (Narea) in their responses to elevated O3, and the allocation of N to different leaf components: photosynthetic apparatus, cell walls, and others under elevated O3 level. Our results showed that elevated O3 significantly increased Nmass, but reduced Narea and leaf mass per area (LMA). The two clones showed no difference in Nmass response to O3, but the more sensitive clone showed greater reduction of Narea and LMA due to O3. We also found positive relationships between Narea and photosynthetic parameters, e.g. light-saturated photosynthetic rate (Asat). Furthermore, elevated O3 significantly reduced photosynthetic N-use efficiency (PNUE) and leaf N allocation to photosynthetic components, while increasing N allocation to cell walls and other components. We concluded that plants invested more N in cell walls and other components to resist O3 damages at the expense of photosynthetic N. The change of N allocation in plant leaves in response to elevated O3 could have an impact on ecological processes, e.g. leaf litter decomposition. © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian District, Beijing 100085, China. E-mail address: [email protected] (Z. Feng).

https://doi.org/10.1016/j.scitotenv.2018.11.471 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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1. Introduction Ozone (O3) is an important phytotoxic air pollutant generated by photochemical reactions of the precursors including volatile organic compounds, nitrogen oxides, carbon monoxide and methane (Simpson et al., 2014). The increased emissions of O3 precursors, have raised tropospheric O3 concentrations globally since the industrial revolution, and will continue to do so in various regions of the world in the future (Young et al., 2013). China is one of the regions where increased surface O3 concentration has become a major environmental pollution (Feng et al., 2015). According to Wang et al. (2017), O3 levels exceeded the ambient air quality standard of China (hourly and the maximum 8hour values are 200 μg/m3 and 160 μg/m3, respectively, http://www. mep.gov.cn) by 100–200% in urbanized regions of China, such as the Yangtze River delta, Jing-Jin-Ji region, and the Pearl River delta. The high O3 concentrations in these regions have caused various damages in plants such as visible foliar injury, premature senescence, reduction in photosynthesis, stomatal closure, and chlorophyll degradation (e.g. Gao et al., 2017; Li et al., 2017; Xu et al., 2018). We focus in this study on the effects of O3 on leaf nitrogen (N), which is among the major constituents of plants mainly existing in proteins, nucleic acids and chlorophylls (Luo et al., 2013). Its concentration is strongly related with photosynthetic rate and other photosynthetic parameters (e.g., the electron transport rate and the carboxylation capacity) (Hikosaka, 2004). Ozone damages leaf tissues and impairs carbon (C) fixation (e.g., Matyssek and Sandermann, 2003; Yendrek et al., 2017), which then affects the belowground parts: roots and mycorrhizae and alter soil processes (e.g., Andrew et al., 2014; Grulke et al., 1998; Kasurinen et al., 1999; Wu et al., 2016). The adverse effects on belowground parts could also hinder the uptake and allocation of N (e.g., Cao et al., 2016; Luedemann et al., 2005; Weigt et al., 2015). There have indeed been many studies on the effects of O3 on leaf N concentration, but their results are inconsistent with each other. Elevated O3 increased leaf N concentration (e.g., Cao et al., 2016; Shang et al., 2018), had no significant effects (e.g., Häikiö et al., 2006; Oksanen et al., 2005), or reduced it (e.g., Lindroth et al., 2001; Singh et al., 2009). The past studies are also inconsistent in their reported units of the leaf N concentration, which was expressed on leaf mass basis in some studies (e.g., Oksanen et al., 2005; Shang et al., 2018), but was based on per unit leaf area in other studies (e.g., Kitao et al., 2009; Oikawa and Ainsworth, 2016). In some studies, both massbased and area-based leaf N concentrations were reported (Hoshika et al., 2013; Koike et al., 2012; Watanabe et al., 2013, 2018). Since different units of physiological indicators reflect different emphasis on physiological processes (Hikosaka, 2004), it is necessary to test whether the effects of O3 on leaf N differ between the mass-based (Nmass) and areabased (Narea) concentration. About half of the total leaf N is used for photosynthesis being allocated to three main parts: Rubisco, bioenergetics and light-harvesting proteins (Hikosaka and Terashima, 1995; Takashima et al., 2004). Small changes of photosynthetic N can affect the CO2 assimilation rate under light-saturated conditions (Asat) and photosynthetic nitrogenuse efficiency (PNUE) of plants (Feng et al., 2009; Onoda et al., 2004). In addition to photosynthetic apparatus, cell walls also act as a major N sink in leaves (Lambers and Poorter, 1992), where the main function of N is for plant defense (Lambers and Poorter, 1992; Showalter, 1993; Feng et al., 2009). Some studies have focused on the trade-off of leaf N allocation between the different components (e.g., Feng et al., 2009; Onoda et al., 2004; Takashima et al., 2004). For example, Takashima et al. (2004) found that the N allocation of the leaf to the photosynthetic apparatus was greater in deciduous species than those in evergreen species, but that the N allocation to cell walls was smaller in the former species. Leaf N allocation was also different between early germinating and late germinating plants. Those that germinated later had a significantly larger allocation of N to photosynthesis than those that germinated

earlier, which was associated with a smaller allocation of N to cell walls in late germinators (Onoda et al., 2004). Feng et al. (2009) showed that a noxious invasive plant throughout the subtropics, Ageratina adenophora, had experienced selection for reduced N allocation to cell walls for defense and increased N allocation to photosynthesis for growth. There were also reports on leaf N allocation under global changes, as indicated by the fact that elevated CO2 and N addition increased allocation of leaf N to photosynthesis and reduced one to cell walls (Zhang et al., 2016). Despite the studies on N allocation in different organs of whole plants under elevated O3 (Weigt et al., 2015), few have focused on the N allocation in plant leaves (Watanabe et al., 2013), especially cell wall N, under elevated O3 concentration. As many studies have confirmed that O3 induced negative effects on photosynthesis and photosynthetic enzymes (e.g. Rubisco) of plants (Feng et al., 2011; Häikiö et al., 2009; Noormets et al., 2001; Shang et al., 2017), elevated O3 could reduce the allocation of N to photosynthesis (Watanabe et al., 2013). We therefore hypothesized that plants invest more N in cell walls to protect against O3 damages at the expense of photosynthetic N, and tested the hypothesis with poplar under elevated O3. The objectives of the present study were (1) to determine O3 exposure-response relationships with Nmass and Narea of poplar; and (2) to reveal the leaf N allocation to photosynthesis (growth) and cell walls (defense) in poplar leaves under elevated O3 concentration. 2. Materials and methods 2.1. Experiment site and plant materials We conducted the experiments at a rural site of Yanqing, northwest of Beijing (40°30′N, 116°E) in 2016 and 2017. The study area is characterized by a continental monsoon climate type. The experiment in 2016 was conducted to determine O3 exposure-leaf N response relationships in two widely-planted hybrid poplar clones ‘546’ (P. deltoides cv. ‘55/56’ × P. deltoides cv. ‘Imperial’) and ‘107’ (P. euramericana cv. ‘74/76’). Our previous studies (Hu et al., 2015; Shang et al., 2017) have shown that the two clone have similar leaf morphology and phenology, but that clone 546 is more sensitive to O3 than clone 107. The experiment in 2017 was conducted to investigate the effect of elevated O3 concentration on leaf N allocation to photosynthetic apparatus and cell walls in the O3-sensitive clone 546. In both 2016 and 2017 experiments, rooted cuttings of hybrid poplar clones were planted in 1-L peat containers in April. When the cuttings grew to about 10 leaves, they were transplanted into 20 L circular plastic pots filled with farmland soil from 0 to 10 cm depth, sieved out by a 0.3 cm pore mesh and then carefully mixed to achieve a homogeneous mixture. The concentrations of NH4-N and NO3-N in the mixed soil were 6.5 ± 2.2 and 4.6 ± 1.4 mg N kg−1 dry soil, respectively. The potted plants were grown in the field for about two months and those with similar height and base diameter were subjected to the O3 treatments. 2.2. Ozone treatments The O3 fumigation was conducted in open-top champers (OTCs), which had an octagonal base (12.5 m2 of growth space and 3.0 m of height) and was covered with fortified glass. The experiment in 2016 had five O3 treatments: charcoal-filtered ambient air (CF), non-filtered ambient air (NF), and NF with targeted O3 addition of 20 ppb (NF20), 40 ppb (NF40), and 60 ppb (NF60) in 15 OTCs. In 2017 experiment, however, 6 OTCs were used for two O3 treatments: CF and NF40. Each treatment used three OTC replicates. The monitoring and control of O3 concentration within OTCs were described in Shang et al. (2017) in detail. The plants were grown for about 10 days for acclimatization to the chamber environment before being subjected to the O3 fumigation for 96 days from 26 June to 30 September in 2016 and 105 days from 10

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June to 22 September in 2017. Fumigation was done from 08:00 to 18:00 when there was no fog, mist, rain, or dew, and the daily maximum fumigation period was 10 h. During the experiment, the plants were fully watered to avoid drought stress. The values of mean O3 concentrations and AOT40 (accumulated O3 exposure over an hourly threshold of 40 ppb) during the experiment in 2016 and 2017 are shown in Table 1. The average air temperature and relative humidity inside the OTCs were 23.7 °C and 80.4% during the O3 fumigation in 2016, and were 22.4 °C and 83% during the O3 fumigation in 2017, respectively. 2.3. Parameter measurements and calculations 2.3.1. Gas exchange measurement A portable photosynthetic system fitted with a 6400-40 leaf chamber fluorometer (LI-6400XT, LICOR Corp, USA) was used to determine gas exchange parameters of a leaf at middle position (11-13th fully expanded leaves from the top) from each plant. This study had two datasets of gas exchange parameters. In 2016 experiment, Asat was measured on 8 & 9 September for clones 546 and 107, respectively. Two plants were randomly selected for the measurement in each OTC. During the measurements of Asat, the photosynthetic photon flux density (PPFD) was set at 1200 μmol m−2 s−1, CO2 levels at 400 ppm, block temperature at 30 °C, and relative humidity (RH) between 45% and 55%. Mass-based light-saturated rate of CO2 assimilation (Amass) was calculated by Asat and leaf mass per area (LMA). On the other hand, in 2017 experiment with clone 546, the A-Ci curve was generated by the automatic program in the LI-6400 photosynthesis system in July and September. Two plants were randomly selected for measurement in each OTC. During the A-Ci measurements, the PPFD was kept at 1200 μmol m−2 s−1, the block temperature was set at prevailing environmental conditions (25–30 °C, the leaf temperature as 23–32 °C) and relative humidity was kept at 50–60%. The ambient CO2 concentration (Ca) was adjusted in a series of 400, 300, 250, 200, 150, 100, 50, 400, 500, 700, 1000, 1300, 1600 ppm. During each step of CO2 change, the minimum and the maximum waiting times were 4 and 6 min, respectively. The maximum rate of electron transport (Jmax) and the maximum carboxylation efficiency (Vcmax) were determined following the Farquhar, von Caemmerer, and Berry model (FvCBmodel; Farquhar et al., 1980; von Caemmerer and Farquhar, 1981). 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). The parameters: Vcmax and Jmax were used to calculate N allocation to photosynthesis (Rubisco and bioenergetics). 2.3.2. Chlorophyll content After the gas exchange parameter measurements, two leaf discs of 0.7 cm diameter from per plant for chlorophyll were extracted in 2 mL 95% ethanol at 4 °C for 72 h in the dark until totally fading. The extracts were then measured for chlorophyll content (Chl) by the specific absorption coefficients of Lichtenthaler (1987). Mass-based chlorophyll content (Chlmass) was calculated by Chl and LMA. The Chl was used for

Table 1 The values (mean ± SD) of mean daytime ozone concentrations and AOT40 (accumulated O3 exposure over an hourly threshold of 40 ppb) during the experimental period in 2016 (from 26 June to 30 September, 96 days) and 2017 (from 10 June to 22 September, 105 days). Years

Treatments

10-h average O3 (8:00–18:00) (ppb)

AOT40 (ppm h)

2016

CF NF NF20 NF40 NF60 CF NF40

24.85 ± 1.46 45.17 ± 0.57 60.37 ± 1.77 75.71 ± 1.39 89.89 ± 0.89 24.03 ± 0.79 80.62 ± 2.68

2.48 ± 0.45 11.65 ± 0.38 22.77 ± 1.38 36.90 ± 1.37 50.83 ± 0.79 2.44 ± 0.40 41.63 ± 2.62

2017

171

the calculation of N allocation to the light-harvesting complex and photosystems. 2.3.3. Leaf mass per area After the gas exchange parameters measurements, five leaf discs of 1.2 cm diameter were collected and dried until constant weight at 80 °C from the leaf which had been used to measure gas exchange parameters, and then the dry mass of the discs was measured. LMA was obtained as dry mass (g) divided by leaf surface area (m2) and was used for conversion between area-based and mass-based indicators. 2.3.4. Total nitrogen concentration A half leaf that was previously used to determine gas exchange parameters was collected from each plant for N concentration, and then were dried at 80 °C for 72 h to constant mass and ground with a ball mill. Nmass was determined with an elemental analyzer (Vario EL III, Elementar, Germany). Narea (g m−2) was obtained from Nmass (mg g−1) and LMA (g m−2) as: Narea ¼ Nmass  LMA  1000

ð1Þ

2.3.5. Photosynthetic nitrogen-use efficiency (PNUE) PNUE (μmol g−1 N s−1) was calculated from Asat (μmol m−2 s−1) and Narea (g m−2) as: PNUE ¼ Asat =Narea

ð2Þ

2.3.6. Estimation of nitrogen allocation to photosynthesis The photosynthetic apparatus of plants was divided into three parts: (1) Rubisco, (2) bioenergetics (electron carriers except for coupling factor, photosystems and Calvin cycle enzymes except Rubisco), and (3) light-harvesting complex and photosystems (Hikosaka and Terashima, 1995; Takashima et al., 2004). The N concentrations in Rubisco, bioenergetics and light-harvesting complex were expressed as NR (g m−2), NB (g m−2) and NL (g m−2), respectively. NR (g m−2) was obtained according to the equation (Niinemets et al., 1999; Watanabe et al., 2012): NR ¼ Vcmax =ð6:25Vcr Þ

ð3Þ

where Vcr is the specific activity of Rubisco (the maximum rate of RuBP carboxylation per unit Rubisco protein). The factor of 6.25 (g Rubisco [g N in Rubisco]−1) converts N content to protein content. Vcr is assumed to be 20.5 mol CO2 (g Rubisco)−1 s−1 for purified Rubisco enzyme at 25 °C from Spinacia oleracea (Jordan and Ogren, 1984). NB (g m−2) was estimated according to the following equation (Takashima et al., 2004): NB ¼ Jmax =ð156  9:53Þ

ð4Þ

We assumed that N in bioenergetics is proportional to Jmax, where the ratio of Jmax to the cytochrome f content is 156 mmol mol−1 s−1 (Niinemets and Tenhunen, 1997) and N in bioenergetics per unit cytochrome f is 9.53 mol mmol−1 (Hikosaka and Terashima, 1995). NL (g m−2) was calculated with the following equation: NL ¼ 37:1Chl

ð5Þ

where Chl (mol m−2) and the N content per unit Chl is 37.1 mol mol−1 (Evans and Seemann, 1989). The total N concentration for photosynthesis (NP, g m−2) was calculated as the sum of the three parts above: NP ¼ NR þ NB þ NL

ð6Þ

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The percentage of the total leaf N allocated to Rubisco (PR, %), bioenergetics (PB, %), light-harvesting complex (PL, %) and photosynthesis (PP, %) were calculated as:

3. Results

PR ¼ NR =Narea  100

ð7Þ

Results of the 2016 experiment with two clones are presented in 3.1 and 3.2, whereas those of the 2017 experiment with clone 546 are presented in 3.3, 3.4, and 3.5.

PB ¼ NB =Narea  100

ð8Þ

3.1. Ozone exposure-response relationships with Nmass, Narea LMA and PNUE

PL ¼ NL =Narea  100

ð9Þ

PP ¼ NP =Narea  100

ð10Þ

2.3.7. Estimation of nitrogen allocation to cell walls After measurements of gas exchange, the other half leaf was frozen quickly with liquid N and then stored at −80 °C in freezer. N allocation to cell walls was analyzed for the samples according to Takashima et al. (2004) and Onoda et al. (2004). The leaf was homogenized in 1 mL of the phosphate buffer (pH 7.5) (100 mM Na-phosphate buffer with 2 mM MgCl2, 0.4 M sorbitol, 5 mM iodo acetate, 5 mM phenyl methyl sulfonyl fluoride, 10 mM NaCl, 1% polyvinylpyrrolidone, and 5 mM dithiothreitol). The mortar was washed with 3 mL phosphate buffer, which was added to the homogenate. The homogenate was centrifuged at 15,000g for 30 min and the supernatant was discarded. The phosphate buffer containing 3% SDS was added to the pellet and heated at 90 °C for 5 min. The mixture was centrifuged at 4500g for 10 min. This procedure was repeated four times. The final pellet regarded as the SDS-insoluble fraction, were washed with distilled water and ethanol in turn by centrifugation (4500g for 10 min), and oven dried at 75 °C for 2 days. After drying, these pellets were weighed and N concentrations were determined using above-mentioned elemental analyzer. This part of N was considered to be cell walls N (NCW, g m−2), although it might be slightly overestimated because the final pellet contained a small contamination by cytoplasmic proteins (Onoda et al., 2004). The percentage of the leaf N allocated to cell walls (PCW, %) and other N (Pother, except for PP and PCW) was calculated as: PCW ¼ NCW =Narea  100

ð11Þ

Pother ¼ 100−PP −PCW ¼ 100−PR −PB −PL −PCW

ð12Þ

Significant positive correlations were found between Nmass and AOT40 for the two clones (clone 546: P = 0.0013; clone 107: P = 0.0002) whereas Narea and LMA were negatively correlated with AOT40 (Fig. 1). PNUE was also significantly reduced by elevated O3 (Fig. 2). The slope of the exposure response relationships was significantly less in clone 107 than that in clone 546 for Narea (ANCOVA, P = 0.031) and LMA (ANCOVA, P = 0.003) but the two clones did not differ in their response to AOT40 for Nmass (ANCOVA, P = 0.425) or PNUE (ANCOVA, P = 0.382). 3.2. Relationships between total nitrogen concentration and photosynthetic parameters The Nmass was negatively correlated with mass-based photosynthetic parameters for two clones, which showed no difference in the slope of relationships for Amass (P = 0.892), Chlmass (P = 0.311) or PNUE (P = 0.711) (Fig. 3a, c, e). By comparison, Narea was positively correlated with between Asat (P b 0.001), Chl (P = 0.002) and PNUE (P = 0.0032) (Fig. 3b, d, f), with no significant difference between the two clones for Asat (P = 0.471), Chl (P = 0.551) or PNUE (P = 0.558). 3.3. Effects of ozone on partitioning of leaf nitrogen to photosynthesis NR, NL and NP were significantly higher in July than September by 35.3%, 36.9% and 45.2%, respectively, on average across the O3 treatments (Fig. 4a, c, d), whereas NB showed no significant difference between July and September (Fig. 4b). Elevated O3 significantly reduced NR (P = 0.0009), NL (P = 0.0044) and NP (P = 0.0008) but NB (P = 0.3925) (Fig. 4). NF40 significantly reduced NR by 21.8% and 35% relative to CF in July and September for clone 546, respectively. Relative to CF, NB was decreased by 13.7% in NF40 in September, but this was not statistically significant (Fig. 4b). Compared to CF, NF40 significantly reduced NL by 36.2% in July. NF40 also significantly reduced NP by 24.9% and 29.8% in July and September, respectively. On the other hand, the interaction between O3 and time had no significant effects on the N allocation to each part of photosynthesis.

2.4. Statistical analyses 3.4. Effects of ozone on partitioning of leaf nitrogen to cell walls We established O3 exposure (AOT40)-response relationships by linearly regressing the dependent variable: either Nmass, Narea, LMA and PNUE, on AOT40 for two clones in 2016 experiment. The AOT40 value up to the measurement date was 2.11 ppm h, 10.0 ppm h, 18.9 ppm h, 29.9 ppm h and 40.7 ppm h in CF, NF, NF20, NF40 and NF60, respectively. We also estimated relationships between the total N concentration (either Nmass or Narea) and Asat, Chl, or PNUE for the 2016 experiment, and difference in the slopes between the clones was determined by analysis of covariance (ANCOVA) using SPSS18.0 (SPSS 18.0, Chicago, IL, USA). The data of two plants in each OTC were averaged, and single OTC is used as the statistical unit. The data of N allocation from 2017 experiment were subjected to two-way analysis of variance (ANOVA) to test the effects of time (July and September), O3 treatments (CF and NF40) and their interaction on NR, NB, NL, NP, NCW, PR, PB, PL, PP, PCW and Pother for clone 546. The significant differences were identified with P b 0.05 by the Tukey's Honestly Significant Difference (HSD) test. The JMP software (SAS Institute, Cary, NC, USA) was used for the two-way ANOVA. The data of two plants in each OTC were averaged, and single OTC is used as the statistical unit.

Cell walls mass per area was 21.9% higher in September than in July when averaged across two O3 treatments (Fig. 5a). NCW showed no significant difference between July and September (Fig. 5b). NF40 significantly increased NCW by 26.2% relative to CF in July, but had no significant effect on NCW in September (Fig. 5b). Elevated O3 significantly reduced cell wall mass per leaf area by 31.0% and 34.6% in July and September, respectively. 3.5. Effects of ozone on percentage of leaf nitrogen allocated to photosynthesis, cell walls and others Percentage of the total leaf N allocated to each part was not significantly different between the two sampling times except for PB and PCW (Table 2). PCW was 5.1% higher in September than in July when averaged across the two O3 treatments. Elevated O3 had significant effects on percentages of total leaf N allocated to each component except for PB with no significant interaction between O3 and time (Table 2). NF40 relative to CF significantly reduced PP by 15%, and increased PCW and Pother

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Fig. 1. Ozone exposure-response relationships with mass-based nitrogen concentration (Nmass), area-based nitrogen concentration (Narea), and leaf mas per area (LMA) for poplar clone 546 and clone 107 at September in 2016. Each point represents a chamber mean. The results of LMA (e, f) have been presented in Shang et al. (2017). Each point represents a chamber mean.

by 2.2% and 12.9%, respectively, on average across the two samplings times (Fig. 6). 4. Discussion 4.1. Ozone exposure-response relationships in nitrogen concentration Previous studies on O3 exposure-response relationships mostly focused on tree biomass (e.g., Büker et al., 2015; Gao et al., 2017; Hu et al., 2015), while few have focused on physiological variables (Bagard et al., 2015; Shang et al., 2017; Xu et al., 2018). Physiological parameters response is quickly to environmental changes, and the response of foliar physiological traits can be easily detected in the field. In O3 impacts also, physiological variables are more sensitive than biomass, and can be used as an early indicator of the risk of high O3 level (Bagard et al., 2015; Shang et al., 2017). Furthermore, some physiological indicators can be determined easily and non-destructively unlike tree biomass (Xu et al., 2018). Physiological indicators could be estimated with sensors at canopy level and remote sensing for large-scale assessment of O3 risks through exposure-response relationships of the physiological parameters (Chi et al., 2016). In this study, we established exposure-response relationships between AOT40 and the N concentrations for two clones (Fig. 1), also found close relationships between the N concentrations and the photosynthetic indicators (Fig. 3). These

relationships would facilitate the assessment of O3 impacts on C and N cycles in ecosystems. It is a common practice to define the critical levels (CLs) for protection of vegetation against O3 with plant biomass (Büker et al., 2015; Hu et al., 2015; Shang et al., 2017). A previous study indicated the CL based on exposure (AOT40)-response relationship at 14.8 ppm h for a 5% reduction in plant biomass in the same two poplar clones as were studied here (Shang et al., 2017). With the AOT40 value of 14.8 ppm h, the reduction of Narea is 6.83%, which is not significantly different from the 5% reduction in biomass. The significant relationship between Narea and Chl (Fig. 3d) suggests a possibility to estimate Narea easily with a Chl meter, e.g., SPAD (Chl and SPAD can be transformed through a nonlinear function, Uddling et al., 2007). In the nature, leaf N concentration estimated by SPAD can be used as an earlier indicator of plant damage by O3. Furthermore, the slope of the regression line of Narea for clone 107 was smaller than for clone 546 (ANCOVA, P = 0.031) (Fig. 1c, d), which is consistent with previous results showing the higher sensitivity in clone 546 than clone 107 to O3 (Shang et al., 2017). 4.2. Effects of elevated ozone on leaf nitrogen concentration In this study, an interesting result was that the effects of O3 on Nmass and Narea were opposite: O3 significantly increased Nmass of poplar leaves but significantly reduced Narea (Fig. 1). This is due to the fact

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with a net result of higher N concentration (enrichment effect) (Wittig et al., 2009). However, Couture et al. (2017) reported lower birch Nmass in elevated O3 due to inhibition of Rubisco synthesis by O3. Ozone fumigation also affected N metabolism by decreasing nitrate reductase activity in leaves, and reduced mass-based concentrations of nitrate N and ammonium N (Huang et al., 2012). The inconsistent results of leaf N concentration response to O3 could be due to the difference between the studies in O3 treatments, nitrogen economy strategies of the species of concern, e.g., ruderal, oligotrophic and mesotrophic species, phenological stages, and environmental conditions, e.g. nutrition and water status of plants. Different life form of plant species, e.g. deciduous/broadleaf, evergreen/coniferous, woody/herbaceous, perennial/annual species, might have also contributed to the difference among studies. For example, the effect of O3 on plant N metabolism was different under different soil N loads (Yamaguchi et al., 2010). Hoshika et al. (2013) also found that the effect of elevated O3 on N concentration differed between early and late germinated leaves of white birch. The effect of O3 on leaf Nmass would affect the decomposition of litter (Liu et al., 2009), which could alter ecosystem production through nutrient cycling. 4.3. Leaf nitrogen allocation to photosynthesis and cell walls under elevated ozone

Fig. 2. Ozone exposure-response relationships with photosynthetic nitrogen-use efficiency (PNUE) for poplar clone 546 and clone 107 at September in 2016. Each point represents a chamber mean.

that O3 significantly reduced LMA of poplar (Fig. 1), resulting in a decrease in N concentration per unit area. The apparent inconsistency in the reported response of leaf N concentration to elevated O3 might be due to the use of different units of N concentration, in which some researchers reported mass-based N while others reported area-based one (Kitao et al., 2009; Oikawa and Ainsworth, 2016; Oksanen et al., 2005; Watanabe et al., 2013, 2018). Area-based parameters are important for the study of photosynthesis-related processes like CO2 diffusion, light interception and transpiration, as they are measured as a flux per unit leaf area (Hikosaka, 2004). The reduction of leaf Narea by O3 is consistent to our previous results that O3 significantly reduced photosynthetic parameters (Asat, Chl and actual photochemical efficiency of PSII in the light) (Shang et al., 2017). In this study also, Narea was positively related with photosynthetic parameters (Asat, Chl and PNUE) for both poplar clones (Fig. 3). The effect of O3 on leaf Narea directly affects plant's biomass and the ecosystem's carbon sequestration. For leaf economy, however, mass-based parameters are important, because leaf mass represents the investment of biomass for carbon fixation (Hikosaka, 2004). Nmass showed an opposite trend than Narea, and exhibited negative relationships with photosynthetic parameters (Fig. 3). Therefore, it is necessary to select the appropriate unit of leaf N concentration according to the objectives of the research. Interestingly, different responses to O3 have been reported even with the same unit of leaf N concentration. For example, Cao et al. (2016) found increased Nmass in P. bournei and P. zhennan with elevated O3, and interpreted this response as an adaptive strategy of plants for enhanced defense capability against O3 pollution. A meta-analysis also showed a consistent increased Nmass, which was explained by the greater reduction of plant biomass accumulation than N accumulation

Photosynthetic rate of plants is often co-limited by biochemical and stomatal factors under elevated O3. The biochemical limitations mainly include: chlorophyll degradation, impaired carboxylation capacity, reduced electron transport rate and changed endogenous antioxidant metabolism (Feng et al., 2011, 2016; Gao et al., 2016; Zhang et al., 2014). A meta-analysis of the responses of woody species to O3 in China revealed that elevated O3 concentration (109 ppb) significantly reduced Asat by 28% (Li et al., 2017) inhibiting photosynthetic processes of the plants. However, leaf N is inextricably linked with photosynthetic processes. Dividing leaf N for photosynthesis into three fractions, we found in this study that elevated O3 reduced N concentration in the photosynthetic apparatuses (NR, NB and NL), although the reduction of NB was statistically nonsignificant (Fig. 4). Furthermore, NF40 significantly reduced NP by 29.8% relative to CF for clone 546 in September (Fig. 4d). Lower N concentration in the photosynthetic apparatus was associated with a lower PNUE (Takashima et al., 2004). Plants with a high PNUE are related with high productivity habitats and high growth rates, while a low PNUE reflects growth on stressful or low productivity conditions (Hikosaka, 2004). As photosynthetic capacity per unit leaf N, the PNUE is an important leaf trait to reflect leaf economics, strategy and physiology. In the present study, elevated O3 significantly reduced the PNUE (Fig. 2), which further proved that the plants had allocated less N to photosynthetic apparatus under high O3 concentration (Onoda et al., 2004). At the same time, elevated O3 significantly reduced the percentage of total leaf N allocated to photosynthesis (Fig. 6). These results all suggested that elevated O3 reduced leaf N allocation to photosynthesis and less N was used for growth of plants under elevated O3. As an important morphological indicator, LMA can reflect the sensitivity of plants to O3, and leaves with higher LMA are more tolerant to O3 (Feng et al., 2018; Li et al., 2016). Elevated O3 significantly reduced LMA in this study (Fig. 1e, f). Oksanen et al. (2005) have also reported elevated O3 led to thinner leaves. Notably, LMA is positively correlated with structural toughness, and it can be used as an indicator to reflect structural (cell walls) biomass (Reich et al., 1991; Wright and Cannon, 2001). Consistent with lower LMA, cell walls mass per area of leaves in NF40 were lower by 31% and 34.6% relative to CF in July and September, respectively (Fig. 5a). Cell walls are a part of the plant apoplast, which is an important line of defense against O3 in plants (Keutgen and Pawelzik, 2008). Cell walls are also an important N sink (Lambers and Poorter, 1992), as they contain different kinds of proteins, including arabinogalactan proteins, proline-rich proteins, hydroxyproline-rich proteins and glycine-rich proteins (Showalter, 1993; Carpita and

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Fig. 3. Mass-based or area-based light-saturated rate of CO2 assimilation (Amass, Asat) (a, b), Mass-based or area-based chlorophyll content (Chlmass, Chl) (c, d), and photosynthetic nitrogen-use efficiency (PNUE) (e, f) as a function of mass-based nitrogen concentration (Nmass) and area-based nitrogen concentration (Narea), respectively, for poplar clone 546 and clone 107 at September in 2016. Each point represents a chamber mean.

McCann, 2000). In this study, elevated O3 increased the NCW, and the effect was statistically significant in July (Fig. 5b). This is consistent with previous studies showing that the content of cell walls components 0.6

0.08

Time: 0.0001 Ozone: 0.0009 Time × Ozone: 0.7773

(a) a

0.5

(cellulose, lignin and wall-bound ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid)) were increased by O3 fumigation and may contribute to a more pronounced resistance against stress

0.06 -2

NB (g m )

b

-2

NR (g m )

b 0.4

Time: 0.1150 Ozone: 0.3925 Time × Ozone: 0.3718

(b)

0.3

c

0.04

0.2 0.02 0.1 0.0

0.00 Time: 0.0109 Ozone: 0.0044 Time × Ozone: 0.1754

0.3

b b

0.2

(d)

Time: 0.0003 Ozone: 0.0008 Time × Ozone: 0.6778

a

b

0.6

b

-2

-2

NL (g m )

a

0.8

NP (g m )

(c)

b

c 0.4

0.1 0.2 July September 0.0

0.0 CF

NF40

CF

NF40

Fig. 4. Effects of ozone (charcoal-filtered ambient air (CF) and non-filtered ambient air with targeted O3 addition of 40 ppb (NF40)) on the concentration of nitrogen allocation to Rubisco (NR, a), bioenergetics (NB, b), light-harvesting complex and photosystems (NL, c), and photosynthesis (NP, d) for poplar clone 546 at July and September in 2017. Different letters indicate significant differences among bars within each variable (mean ± SD, Tukey test, P b 0.05, n = 3).

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Fig. 6. Effects of ozone (charcoal-filtered ambient air (CF) and non-filtered ambient air with targeted O3 addition of 40 ppb (NF40)) on the percentage of the total leaf nitrogen allocated to Rubisco (PR), bioenergetics (PB), light-harvesting complex (PL) and photosystems (PP), cell wall (PCW), and other (Pother) in leaves for poplar clone 546 at July and September in 2017 (n = 3).

Fig. 5. Effects of ozone (charcoal-filtered ambient air (CF) and non-filtered ambient air with targeted O3 addition of 40 ppb (NF40)) on cell walls mass per area (a) and the concentration of nitrogen allocation to cell walls (NCW, b) for poplar clone 546 at July and September in 2017. Different letters indicate significant differences among bars within each variable (mean ± SD, Tukey test, P b 0.05, n = 3).

(Olbrich et al., 2010). Furthermore, elevated O3 significantly increased PCW, and the PCW in September was higher than in July (Fig. 6; Table 2). These results strongly support our hypothesis that plants allocated more N to the cell walls to resist O3 damage. In addition to the changes in two leaf N components (PP and PCW), elevated O3 also induced a significant increase in Pother (Fig. 6; Table 2). The other N including defensive compounds and nucleic acid (Onoda et al., 2004) is correlated with the content of a series of antioxidant pigments and antioxidant enzymes in the leaves (Liu et al., 2007). The increase of this component of N is consistent with other research results, suggesting that elevated O3 increases some secondary N compounds and antioxidant enzymes in order to enhance the antioxidant capacity of plants. For example, Dai et al. (2017) found that some antioxidant enzyme activities (CAT, SOD and APX) were significantly increased under elevated O3 among 13 peach cultivars. Consistently also, the number of mitochondria and peroxisomes which play an important role in defense, was increased under elevated O3, and it is

known that the peroxisomes contain catalase-enzyme (Oksanen et al., 2005). In short, our results support our hypothesis that plants invested more N in cell walls to resist O3 damage at the expense of photosynthetic N. The existence of this trade-off between growth and defense mainly resulted from secondary metabolism and structural reinforcement, which are physiologically constrained in dividing and enlarging cells (Herms and Mattson, 1992). Further research is necessary to clarify the trade-off strategy between growth and defense from physiological and molecular mechanisms to detoxify O3 pollution. In addition, the N allocation strategy of plant leaves under O3 could have an impact on some ecological processes, for example, the decomposition of leaf litter due to the change of different N components in the leaf.

4.4. Uncertainties In the present study, NR, NB and NL were calculated by using Vcmax, Jmax and Chl, respectively. Although this method has been used in many studies (e.g., Feng et al., 2009; Onoda et al., 2004; Takashima et al., 2004), some of the coefficients in the calculation formulas have uncertainties. For example, the Vcr in Formula (3) was derived from Spinacia oleracea (Jordan and Ogren, 1984), and it might not suit to poplar. Meanwhile, the NCW might be slightly overestimated because it is difficult to completely remove cytoplasmic proteins (Onoda et al., 2004). In addition, poplar saplings were used in the present experiment, which maybe be different from the results of the leaf N allocation in mature trees. Therefore, we need to further verify our results through a long-term O3 fumigation experiment (i.e., O3-FACE systems).

Acknowledgements Table 2 ANOVA results (P values) for main effects and interactions of time (July and September) and ozone (CF and NF40) on the percentage of the total leaf nitrogen allocated to Rubisco (PR), bioenergetics (PB), light-harvesting complex and photosystems (PL), photosynthetic (PP), cell wall (PCW), and other (Pother). Statistically significant effects are marked in bold (P b 0.05, n = 3).

PR PB PL PP PCW Pother

Time

Ozone

Time × ozone

0.2868 0.0109 0.7594 0.8299 b0.0001 0.153

0.0005 0.1194 0.0091 0.0008 0.0013 0.002

0.3893 0.3273 0.4454 0.8448 0.455 0.7513

This study was supported by Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC019), the National Key R&D Program of China (2017YFC0209703) and National Natural Science Foundation of China (41771034). We also express our appreciation to Professor Kazuhiko Kobayashi for English improvement throughout the manuscript. References Andrew, C.J., Diepen, L.T.A.V., Miller, R.M., Lilleskov, E.A., 2014. Aspen-associated mycorrhizal fungal production and respiration as a function of changing CO2, O3 and climatic variables. Fungal Ecol. 10, 70–80.

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