Tropospheric ozone and cadmium do not have interactive effects on growth, photosynthesis and mineral nutrients of Catalpa ovata seedlings in the urban areas of Northeast China

Journal Pre-proofs Tropospheric ozone and cadmium do not have interactive effects on growth, photosynthesis and mineral nutrients of Catalpa ovata seedlings in the urban areas of Northeast China Sheng Xu, Xing-Yuan He, Zhong Du, Wei Chen, Bo Li, Yan Li, Mai-He Li, Marcus Schaub PII: DOI: Reference:

S0048-9697(19)35299-4 https://doi.org/10.1016/j.scitotenv.2019.135307 STOTEN 135307

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Science of the Total Environment

Received Date: Revised Date: Accepted Date:

24 August 2019 14 October 2019 29 October 2019

Please cite this article as: S. Xu, X-Y. He, Z. Du, W. Chen, B. Li, Y. Li, M-H. Li, M. Schaub, Tropospheric ozone and cadmium do not have interactive effects on growth, photosynthesis and mineral nutrients of Catalpa ovata seedlings in the urban areas of Northeast China, Science of the Total Environment (2019), doi: https://doi.org/ 10.1016/j.scitotenv.2019.135307

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Title: Tropospheric ozone and cadmium do not have interactive effects on growth, photosynthesis and mineral nutrients of Catalpa ovata seedlings in the urban areas of Northeast China

Sheng Xua,b, Xing-Yuan Hea,b,c, *, Zhong Dud, *, Wei Chena,b,c, Bo Lia, Yan Lib,c, Mai-He Lie,f, Marcus Schaube,f a

Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, People’s Republic of China

b

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

c

Shenyang Arboretum, Chinese Academy of Sciences, Shenyang 110016, People’s Republic of China

d

College of Land and Resources, China West Normal University, Nanchong 637009, People’s Republic of

China e

Swiss Federal Research Institute WSL, Birmensdorf 8903, Switzerland

f

SwissForestLab, Birmensdorf 8903, Switzerland

* Corresponding authors: Xing-Yuan He, Zhong Du

E-mail addresses: [email protected], [email protected]

Title: Tropospheric ozone and cadmium do not have interactive effects on growth, photosynthesis and mineral nutrients of Catalpa ovata seedlings in the urban areas of Northeast China

Sheng Xua,b, Xing-Yuan Hea,b,c, *, Zhong Dud, *, Wei Chena,b,c, Bo Lia, Yan Lib,c, Mai-He Lie,f, Marcus Schaube,f a

Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, People’s Republic of China

b

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

c

Shenyang Arboretum, Chinese Academy of Sciences, Shenyang 110016, People’s Republic of China

d

College of Land and Resources, China West Normal University, Nanchong 637009, People’s Republic of

China e

Swiss Federal Research Institute WSL, Birmensdorf 8903, Switzerland

f

SwissForestLab, Birmensdorf 8903, Switzerland

* Corresponding authors: Xing-Yuan He, Zhong Du

E-mail addresses: [email protected], [email protected]

Abstract Heavy metal contamination and tropospheric ozone (O3) pollution often co-occur in heavy industrial urban areas, adversely affecting urban plant health. Little is known about the characteristics of growth, physiological metabolism, bioaccumulation of cadmium (Cd) and mineral nutrients in urban trees under the combination of soil Cd contamination and elevated

O3 exposure. In this study, one-year-old street tree Catalpa ovata G. Don seedlings were exposed to Cd contaminated soil (0, 100, 500 mg/kg soil) with 40 µg/m3 O3 (ambient air) and 120 µg/m3 O3 (elevated O3 exposure) for 4 weeks. The results revealed that 500 mg/kg soil Cd addition alone decreased net photosynthetic rate, stomatal conductance, peroxidase activity and increased abscisic acid content and oxidative injury in the leaves of C. ovata. Furthermore, Cd soil contamination decreased leaf, stem, root and total biomass and affected Cd, Mg, Fe, and Zn contents in leaves (P < 0.01), but it did not affect Mg, Fe and Zn contents in roots. O3 exposure did not affect growth, net photosynthetic rate, Cd accumulation and mineral nutrient contents of C. ovata. No interactive effect between Cd and O3 was found on growth, oxidative injury, photosynthetic rate, and the contents of Cd, Mg, Fe and Zn in plant tissues (P > 0.05). Our findings suggest that C. ovata is an appropriate tree species for urban greening and afforestation in heavy industrial urban areas with high O3 pollution in Northeast China. To ensure successful afforestation in heavy industrial areas, the long-term and large scale studies are needed to advance our understanding of the combined effects from extreme climate conditions and multi-pollutant exposure on the metabolism of mature urban trees.

Keywords: Catalpa ovata, Cd contamination, Growth, O3 pollution, Oxidative stress, Physiology, Urban tree

1. Introduction Cadmium (Cd) is one of the most toxic elements that can be regarded as a contaminant in soil (Rizwan et al., 2018). Cd pollution in soil has dramatically increased and has been becoming an increasing environmental issue during recent years, particularly in China (Fan et al., 2016; Yang et al., 2016; Liu et al., 2018; Wei et al., 2019). Soil Cd inhibits plant growth and development by Cd uptake and accumulation within plant organs (Rizwan et al., 2016), affects mineral uptake, photosynthetic rate, antioxidant system, and thereby interferes with the physiological and biochemical processes of plants (Shanmugaraj et al., 2013; Jibril et al., 2017). Among atmospheric pollutants, tropospheric ozone (O3) is considered to be a greenhouse gas with the third strongest radiative forcing on climate (Forster et al., 2007) and most detrimental to plants in Asia (Feng et al., 2014, 2015, 2019; Xu et al., 2015; Li et al., 2016, 2018; Zhang et al., 2018), Europe (Matyssek et al., 2007) and the US (Karnosky et al., 2007). As a strong oxidant, O3 has generally negative impacts on various cellular and molecular processes (Jolivet et al., 2016), leading to altered Rubisco activity (Saxe, 2002), reduced stomatal conductance (Ainsworth et al., 2012), leaf chlorosis and early senescence (Novak et al., 2003; Moura et al., 2018b) and decreased carbon assimilation (Novak et al., 2007; Wittig et al., 2009; Matyssek et al., 2010; Xu et al., 2015). Most agroforestry areas in the vicinity of heavy industrial cities are also impacted by other contaminants besides Cd, leading to combined pollution impacts, which negatively affect sustainable forestry and urban environmental safety (Wang et al., 2018; Xu et al., 2019). Various pollutants may have additive, antagonistic or synergistic interactions on plants (Guo et al., 2012; Ghiani et al., 2014; Marzuoli et al., 2016; Xu et al., 2019). Recently, several studies investigated the physiological impacts of Cd or O3 on plants (Mesnoua et al., 2016; Yang et al., 2018; Guo et al., 2019; Yu et al., 2018; Moura et al., 2018a; Gandin et al., 2019), while only few studies

focused on the effects of their combined exposure on plant physiological metabolism. Most of the studies on the combined effects of O3 and heavy metals focused on crops and only few on woody plants (Li et al., 2010; Guo et al., 2012; Castagna et al., 2013). The combination of O3 and heavy metal exposure was found to reduce photosynthetic capacity, decrease antioxidant enzyme activity, and inhibit plant growth through reducing mineral uptake (Guo et al., 2012; Xu et al., 2019). Further increases of tropospheric O3 exposure and Cd soil contamination are expected to co-occur in heavy industrial cities of China, such as Shenyang known for its old industrial base built in the past decades. Therefore, a better understanding of the combined impacts of Cd and O3 on plants, especially urban trees, is crucial for urban vegetation and afforestation safety in heavy industrial regions (Wang et al., 2018; Xu et al., 2019). Catalpa ovata G. Don is a fast-growing urban tree species with deep growing roots. This tree species is known to be highly tolerant to SO2, smoke, dust deposition, saline and heavy metal contamination (Shan et al., 2011), and therefore widely cultivated and planted as landscape greening and street tree in many heavy industrial cities in northern China (e.g. Shenyang in Northeast China; Li et al., 2013; Zhang et al., 2018). In a previous study, we demonstrated that C. ovata also showed high tolerance of O3 (Xiong et al., 2017). However, so far it is not known how this tree species would grow and exert its physiological metabolism under the combination of elevated O3 exposures and heavy metal soil contamination. In this study, we therefore aim (1) to investigate plant growth and photosynthetic parameters under elevated Cd and/or O3 concentrations; (2) to test combined effects of Cd and elevated O3 on oxidative injury, antioxidative enzyme activity including superoxide dismutase (SOD) and peroxidase (POD), and abscicic acid (ABA) content in leaves; (3) to evaluate the effects of Cd and/or O3 on Cd accumulation and other mineral nutrient (Mg, Fe and Zn) contents in different plant tissues such as leaf, stem and root. In this study, we hypothesize that soil Cd addition can exacerbate the adverse effects of O3 pollution on the tested urban tree species.

The results obtained will enhance our understanding of C. ovata’s physiological tolerance to the combined pollution of Cd soil contamination and elevated O3 exposures.

2. Materials and methods 2.1. Study site and materials The study was conducted in the Shenyang Arboretum (41° 46′ N, 123° 26′ E), which is located close to the densely populated commercial center of Shenyang, China (about 10,000 people per km2). The region is affected by warm temperate-zone semi-humid continental monsoon climate and has an annual average temperature of 7.4 °C (Xu et al., 2014) with a mean January-temperature of -12.6°C and a mean July-temperature of 27.5 °C (Xu et al., 2014). Eighteen one-year-old C. ovata seedlings (70±10 cm in height) were selected from a nearby nursery and transplanted into 9-liter plastic pots filled with 2 kg of soil composed of sand, peat, and clay (2:3:1, v:v:v) on 10 May 2016. All seedlings were potted and grown in a greenhouse with 26/22°C day/night temperature, 65-85% relative humidity (RH), and a 12-h photoperiod at 500 μmol/m2 sec of photosynthetically active radiation (PAR), respectively. The available N, P and K contents in the potting soil was 2.37, 1.78 and 6.24 mg/g, respectively. Soil Cd concentration in the pots was averaged to 0.84 mg/kg potting soil. More detailed information about the physical and chemical soil properties can be found in Xu et al. (2019). The pots were irrigated twice per week until field capacity was reached to ensure sufficient water supply during the growing period in the greenhouse.

2.2. Experimental design

This experiment was conducted in six open-top chambers (OTCs, 4 m in diameter and 3 m in height). Three of them were randomly selected for ambient air (AA, control, 40 µg/m3 O3) and the other three for elevated O3 (EO, 120 ± 10 µg/m3) treatment (Xu et al., 2019). Seedlings were cultivated in pots for 60 days prior to treatments, from 10 May to 10 July 2016. By the end of the cultivation period, the soil in the pots was irrigated with three Cd levels (0, 100 and 500 mg Cd/kg dry soil weight, namely Cd-0, Cd-100, Cd-500) according to the similar studies (Zhang et al., 2008; Miller et al., 2016) and supplied as CdCl2 solution (100 ml) premade by deionized water, with three replicates for each Cd treatment. After Cd pretreatment for one week in the greenhouse, half of the Cd-0-, Cd-100- and Cd-500-pretreated plants were moved into the OTCs with AA, and the other half were moved into OTCs with EO. A transparent rain cap in each OTC was located 0.8 m above all the pots to prevent rainfall. O3 fumigation began on 18 July 2016 and lasted for four weeks. O3 concentrations in each chamber were monitored using an automated time-sharing system connected to an ozone monitor (S-905, Aeroqual New Zealand) and all data were stored using a data logger (CR800, Campbell Scientific Inc., Logan, UT, USA). Target O3 concentration was generated from pure medical oxygen using the high-voltage discharge by the O3 generator (Xinhang-2010, Shenyang, China) and maintained to the relative stable levels by an automatic controller (SDM-CD16AC, Beijing Truwel Instruments, Inc.). O3 was released from 9:00 to 17:00 throughout the experimental period. During the experiment period, the mean and maximum O3 concentrations, and AOT40 (the sum of the differences between the hourly mean ozone concentration in ppb and 40 ppb for each hour of fumigation) after 4 weeks of O3 exposure were recorded and calculated under AA (38.2 and 66.5 µg/m3, 269.8 µg/m3·h) and EO (116.5 and 132.0 µg/m3, 15449.2 µg/m3·h), respectively. 200 ml of deionized water were added to each pot twice a week during O3 exposure. At the end of this experiment, growth and physiological parameters, Cd and mineral nutrient contents in plants

were measured. More detailed information about the experimental design can be found in our previous studies (Xu et al., 2012, 2015, 2017).

2.3. Measurements 2.3.1. Growth All 18 plants in the OTCs were harvested to determine their growth parameters after 4 weeks of treatment. Roots, stems and leaves of each plant were separated and the dry weight (g) was determined after drying at 65 °C until constant weight was reached. 2.3.2. Gas exchange parameters In each OTC, two fully expanded upper leaves per seedling from 3 plants were selected. Leaf gas exchange was measured using a portable Li-Cor 6400 photosynthesis system (Li-6400, Lincoln NE, USA). Net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration (Tr) were measured at 60% RH, 25℃ air temperature, 1,000 μmol/m2 sec PAR, and 400 µmol/mol CO2 concentration from 9:00 to 11:00. 2.3.3. Determination of oxidative injury, antioxidative enzyme activity and ABA content Oxidative injury was assessed by malondialdehyde (MDA) content according to the method of Dhindsa et al. (1981) with minor modifications. The absorbance of thiobarbituric acid reactive substances (TBARS) in the reactive mixture was measured at 532 and 600 nm. The non-specific absorbance at 600 nm was subtracted from the absorbance at 532 nm. The concentration of MDA was calculated using an extinction coefficient of 155 mM/cm. Antioxidative enzyme was extracted according to Cho and Seo (2005) with a slight

modification. Samples of frozen leaves (0.2 g fresh weight, FW) were ground with liquid nitrogen and homogenized in 2.0 ml ice-cold 100 mM phosphate buffer (pH 7.8) containing 0.1 mM EDTA and 1.0% polyvinyl pyrrolidone (PVP). The homogenates were centrifuged at 12,000 g for 20 min at 4℃, and the supernatants were used for the analysis of SOD and POD activities. SOD activity was measured by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Dhindsa et al., 1981). The absorbance was recorded at 560 nm, and one unit of enzyme activity was defined as the amount of enzyme causing 50% inhibition of NBT reduction. POD activity was determined according to the modified method of Chance and Maehly (1955). Reaction mixture (3 ml) was composed of 0.1 M potassium phosphate buffer (pH 7.0), 20 mM guaiacol and 1 mM H2O2. The reaction was initiated by adding 0.02 ml enzyme extract. The increase of absorbance at 470 nm during 1 min was measured. One enzymatic unit (U) was defined as 0.01 absorbance increase per minute, and POD activity expressed as U/g FW. For ABA content, fresh leaf from each treatment was collected, immediately frozen in liquid nitrogen, and then dried in lyophilization. Each sample (0.1 g FW) was ground and extracted with 80% methanol, using 200 ng of ABA as internal standard. Three replicates were performed for each sample. ABA content was measured by the method of enzyme‐linked immunosorbent assay (Yang et al., 2001). ABA concentration was calculated as ng/g FW. 2.3.4. Evaluation of Cd and other mineral element contents in plants After full harvest of the whole plant, the different tissues (leaves, stems and roots) were separated and thoroughly washed with deionized water, and then oven-dried at 65°C to reach constant weight. The dried samples were ground, weighed, and digested with concentrated

HNO3/HClO4 (4:1, v/v). Cd, Mg, Fe and Zn contents in the digested solution were analyzed by atomic absorption spectroscopy (Thermo M6, USA). All the contents in each sample were measured in triplicate. 2.4. Statistical analyses The experiment was set up in a completely randomized split-plot design. Chambers corresponding to the same treatment were considered statistical replicates. There were three replicated OTCs for AA and EO, respectively. One-way analysis of variance (ANOVA) was used to detect significant differences of each parameter between AA and EO with or without Cd addition. The significant differences between the treatments were analyzed by the least significance differences (LSD) at 95% confidence level by using SPSS statistical software (SPSS 18.0, Chicago, USA). Differences between the treatments were considered significant at P < 0.05. The values presented here are the mean of measurements with three replicates (plants) in each set. The individual and interactive effects of Cd and O3 on growth, physiological parameters, Cd accumulation, and mineral nutrient contents in different organs were evaluated by general linear model (GLM).

3. Results 3.1 Effects of Cd and/or O3 on growth of C. ovata seedlings Table 1 shows the biomass accumulation of C. ovata control plants, exposed to Cd and elevated O3 (EO). Compared to the control plant in ambient air (AA) and Cd-0, total biomass and root biomass decreased under Cd-100 and Cd-500. Under EO, leaf, stem and root biomass with Cd-500 significantly decreased by 39.6% (P = 0.015), 51.5% (P = 0.003) and 42.1% (P < 0.05), respectively, in comparison with the control plants. No significant effects

were observed between AA and EO on all growth parameters of plants without Cd addition. General linear model (GLM) analyses revealed that there was no significant effect from O3 × Cd interaction on leaf biomass (P = 0.413), stem biomass (P = 0.675), root weight (P = 0.350), total biomass (P = 0.298) and R/S ratio (P = 0.582), respectively (Table 1). 3.2 Effects of Cd and/or O3 on physiological parameters in leaves of C. ovata seedlings No significant differences for gas exchange parameters were observed between Cd-0 and Cd-100 regardless of O3 fumigation (Fig. 1A-D). Compared to control without Cd addition, Cd-500 significantly decreased Pn, gs and Tr by 93.7%, 69.8% and 84.7% under AA, and by 66.2%, 64.9% and 38.7% under EO, respectively (Fig. 1A, B, D). MDA content in leaves of Cd-100 and Cd-500 polluted plants increased by 2.4 and 5.6 times, 70.3% and 61.4% of the control plants under AA and EO (Fig. 1E), respectively. However, no significant increase in MDA content was found under EO without Cd addition. Furthermore, no significant difference in SOD activity was observed among Cd addition regardless of O3 fumigation (Fig. 1F). POD activity showed a decreasing trend with increasing Cd levels, and decreased by 21.1% and 52.6%, 59.2% and 66.9% compared to the control plants regardless of the O3 fumigation (Fig. 1G). Under EO, ABA content in leaves significantly increased by 71.7% (P<0.05) and 79.4% (P<0.05) under Cd-100 and Cd-500, respectively, compared to that of the control plants (Fig. 1H). 3.3 Effects of Cd and/or O3 on the Cd and mineral nutrients contents in C. ovata seedlings Cd-500 significantly increased the Cd content in leaves and roots compared to the control plants regardless of O3 fumigation (Fig. 2). However, differences in Cd content in all organs

between Cd-100 and Cd-0 were not significant (P > 0.05). GLM analysis revealed that there was no significant interactive effect between Cd and O3 on Cd, Mg, Fe, Zn contents in the different tissues, respectively (P > 0.05, Fig. 2). Similarly, O3 did affect Cd and mineral nutrient concentrations in all organs of C. ovata seedlings. Cd pollution had a significant effect on Mg, Fe, and Zn content in leaves (P < 0.01), but no significant effect on Mg (P = 0.075), Fe (P = 0.983) and Zn (P = 0.143) content in the roots, respectively (Fig. 2).

4. Discussion 4.1 Effect of soil Cd contamination on C. ovata seedlings Cd is known to be highly toxic to plants, and its negative effects on growth have been widely demonstrated for different street tree species such as poplar and willow (Guo et al., 2015; He et al., 2017). In this study, Cd treatments significantly reduced total biomass, particularly in the roots and leaves of C. ovata seedlings. Similar results were reported where Cd inhibited growth of Populus canescens (Dai et al., 2013), Lonicera japonica (Jia et al., 2015), and Koelreuteria paniculata (Yang et al., 2018). However, these trees were exposed to different Cd concentrations and grown under nutrient solutions which might have increased the susceptibility of the plants compared to the current study where no nutrient was added to the soil. Our most recent experiment also showed that high Cd (500 mg/kg) addition in soil decreased root growth, total biomass and root/shoot (R/S) ratio (Xu et al., 2019). R/S ratio is an important index for assessing plant health, particularly under adverse environmental conditions (Agathokleous et al., 2018). In this study, Cd addition in soil slightly decreased R/S ratio with no significant effect, which indicates that C. ovata may be more tolerant to Cd

than native hybrid poplar (Xu et al., 2019). As a strong oxidative pollutant in the soil, Cd can lead to a poisonous effect on the photosynthetic apparatus by root uptake and transfer to the leaves. In the current study, Pn and gs of C. ovata leaves decreased under high Cd soil contamination, indicating that the photosynthetic apparatus has been negatively affected by Cd, reaching a toxic level. Similar results were found that Cd-stressed plants showed the significant photosynthetic inhibition in leaves of tomato (Ahammed et al., 2013) and wheat (Dobrikova et al., 2017). However, lower Cd concentrations in the soil had no significant effect on Pn, which is in agreement with our recent study in a hybrid poplar (Xu et al., 2019). As an abiotic stressor, Cd has generally a negative impact on cell membrane stability of plants (Zhou et al., 2018). In this study, we observed that MDA concentration increased with increasing of Cd levels in soil, suggesting that Cd pollution exacerbated membrane lipid peroxidation and oxidative stress in C. ovata seedlings. It is well known that antioxidant enzymes under Cd stress play an important role in preventing oxidative stress by scavenging reactive oxidative species (ROS) (Qin et al., 2018). In the current study, we found that Cd addition decreased POD activity, in agreement with the result reported by Yang et al. (2018) in leaves of Koelreuteria paniculata seedlings, which may be due to the inhibition of Cd-mediated enzyme synthesis or changes in the accumulation of enzyme subunits caused by ROS (Sidhu et al., 2017). In our study, Cd addition increased SOD activity in C. ovata leaves, as found in rice (Liu et al., 2017) and poplar (Xu et al., 2019). In addition, ABA plays an important role in adaptation to Cd stress during growth and development of plant. Plants can maintain their water balance by closing their stomata, induced by increasing ABA content in leaf tissues (Sharma and Kumar, 2002). The current

study suggests that increasing of ABA content, leading to the reduction in gs and Tr might be a plant reaction to alleviate Cd stress, as described by Chaca et al. (2014). Furthermore, Li et al. (2014) found that ABA accumulation in plants can regulate the antioxidative system to alleviate Cd toxicity. However, an increase in ABA content also implies an aggravation in senescence of plant tissues (Chaca et al., 2014; Yang et al., 2018), as was found in this study where high leaf discoloration rate and early leaf abscission in C. ovata seedlings exposed to high Cd pollution goes along with a significant increase of ABA content in leaf tissues. Similar results were found in Kosteletzkya virginica (Han et al., 2013) and P. alba ‘Berolinensis’ (Xu et al., 2019) that Cd increased the senescing compound ABA concentration in leaf tissue. Cd addition in soil generally leads to increased Cd accumulation and allocation to different plant tissues. Here, increasing Cd contents in plant tissues were correlated to Cd concentrations in the soil. Clemens and Ma (2016) found similar results where increased Cd accumulation was reflecting higher Cd concentrations in the soil. In addition, Cd soil contamination has the potential to affect mineral nutrient cycling and element metabolism of plants. Khaliq et al. (2019) found that mean Cd content in different tissues of rice was from the highest to lowest level in roots, stem, and leaves, respectively, and Cd addition reduced the accumulation of Fe, Zn, and Mg in these tissues. Our experiment indicated that high Cd soil contamination promoted the accumulation of Mg, Fe and Zn, particularly in roots, implying that the equilibrium among mineral elements in plants was disrupted by Cd pollution, which might be one of the major reasons for Cd’s potential toxic impact on plants (Wan et al. 2009). Zorrig et al. (2010) reported that Cd had a detrimental effect on the micro- and macro-element balance in plant tissues and it would display a large

panel of toxic effects once accumulated in plant tissues. 4.2 Effect of elevated O3 exposure on C. ovata seedlings As air pollutant, tropospheric O3 with high oxidative toxicity has in general a negative impact on tree growth and development at seedling stage (Novak et al., 2008; Xu et al., 2015). In our present study, elevated O3 exposure alone decreased growth of C. ovata seedlings after 4 weeks of gas fumigation, but it did not affect Pn and gs. Similar findings were reported by Marzuoli et al. (2016) where elevated O3 had not effect on Pn of Quercus robur young trees, but drained the energy and reduced the power produced by Pn to counteract the O3-induced oxidative stress. As a matter of fact, O3 alone significantly inhibited growth of C. ovata seedlings after 20 days of fumigation by elevated O3 concentration (160 µg/m3) found in our previous experiment (Xiong et al., 2017) but no growth reduction was found in our present study (120 µg/m3). However, despite the growth reduction in the earlier study, Pn and gs of C. ovata leaves showed no significant decline even under 160 µg/m3 O3 (Xiong et al., 2017), which indicates that the photosynthetic apparatus shows a high tolerance to O3 stress by means of detoxification mechanism although there was a slight increase of MDA content in leaves under elevated O3 in this study (Fig. 1E). In fact, elevated O3 exposure can usually induce membrane lipid peroxidation and increase MDA content (Xu et al., 2015; Podda et al., 2019). In this study, O3 fumigation had no significant effect on MDA content in leaves, which implies that no or low oxidative stress occurred until the end of this experiment, most likely resulting from the increasing antioxidant enzyme activity as a protection against O3 injury (Lu et al., 2009; Fu et al., 2015). However, SOD and POD activities decreased under elevated O3

in this study, in agreement with the results found in Pinus halepensis (Scalet et al., 1995) and winter wheat (Liu et al., 2015). Species specific differences in antioxidant enzyme activities may reflect different physiological adjustment capacities in response to elevated O3 (Xu et al., 2015). Activities of SOD and POD might suffer from a complicated fluctuation during plant exposure to O3 fumigation although we had only one time for sampling at the end of this experiment. Similar results were reported where the activities of antioxidant enzymes showed different responses for different enzymes to O3 exposure (Yan et al., 2010; Xu et al., 2017). Furthermore, an increased tolerance towards antioxidative stress may be induced by the accumulation of ABA in plants exposed to O3 (McAdam et al., 2017), as shown in our previous study in leaves of Ginkgo biloba (Li et al., 2011). However, opposite results were found in this study where O3 alone decreased ABA content (P < 0.05). The conflicting findings from our study might be related to species specific differences, differing O3 exposures, and the timing of ABA measurements during O3 exposure. In fact, similar results were reported by Mao et al. (2017) in soybean and Xu et al. (2019) in P. alba ‘Berolinensis’ leaves where ABA content decreased in these plants exposed to elevated O3 concentrations. Under elevated O3, the metabolism of elements in plant tissues may be crucial for growth and health, particularly for protection against O3 stress (Zhuang et al., 2017). Elevated O3 could affect uptake and accumulation of Cd and mineral nutrients in plants (Guo et al., 2012). However, here we found that O3 alone had no significant effect on Cd accumulation in none of the plant tissues of C. ovata, in agreement with the similar studies in hybrid poplar (Xu et al., 2019) and wheat by Guo et al. (2012). Furthermore, we also found that O3 alone had no significant effect on Mg, Fe and Zn within the different plant organs of C. ovata. This is in

agreement with the findings by Zhuang et al. (2017) where elevated O3 concentrations corresponded to only a slight increase of Mg concentrations in the leaves of Phyllostachys edulis. Differing mineral nutrient uptake during O3 exposure implies species specific nutrient utilization among the different plant tissues (Zhuang et al. 2017). For example, Fangmeier et al. (2002) found reduced Mg content in the above-ground but increased Mg content in the below-ground organs of Solanum tuberosum under O3 exposure. 4.3 Combined effect of Cd and O3 exposure on C. ovata seedlings The combined effects of heavy metal and O3 on plants have been reported in many plant species such as wheat (Guo et al., 2012), Lycopersicon esculentum (Degl’innocenti et al., 2014) and poplar clones (Castagna et al., 2013). In this study, no interactive effects were found on growth, Pn, oxidative injury, antioxidant enzyme activity, Cd accumulation, and mineral nutrient contents in C. ovata under the combination of elevated O3 exposure and soil Cd contamination, compared to Cd addition alone under AA. In other words, changes in growth, photosynthetic activity, oxidative injury, Cd accumulation and mineral nutrient uptake induced by the combination of high Cd and elevated O3 concentrations were very similar to those induced by Cd alone under AA, due to the minor effect on plants exposed to elevated O3. Conversely, the results in our recent study indicated that elevated O3 exposures exacerbated Cd accumulation and toxicity of high Cd to O3-sensitive hybrid poplar (Xu et al., 2019). Similar results were found in wheat (Guo et al., 2012). These results indicated that the synergistic effect between Cd and O3 was apt to occur on physiological metabolism of O3-sensitive plants rather than O3-tolerant ones. In conclusion, we found that Cd did not interact with O3 to affect physiology of the tested plant, and high Cd addition significantly decreased growth, photosynthesis, increased membrane lipid peroxidation, oxidative injury and accelerated leaf senescence of C. ovata

rather than elevated O3 exposure. Therefore, we recommend C. ovata as the potentially preferred tree species for urban greening and afforestation in heavy industrial cities in Northeast China exposed to increasing O3 exposure and Cd contaminated soils. Given the limitations of the current study with C. ovata, our findings deepen our understanding of impacts from multifactorial stresses on the physiological adaptation of urban trees and provides a scientific reference for phytoremediation in heavy industrial areas with Cd polluted soils and elevated O3 exposures. However, our new insights and recommendations are based on a short-term study with one-year old seedlings grown under semi-controlled conditions and an extrapolation to mature trees growing under urban conditions must therefore be taken with caution. Numerous studies have demonstrated that varying spatial and temporal scales may alter the results and interpretation from dose-response relationships (e.g., Leuzinger et al., 2011; Cailleret et al., 2018). Furthermore, with progressing climate change, soil water availability may be reduced in future and must therefore take into account for species selection in urban areas and elsewhere. To advance our understanding of the combined impacts of multi-stressors from heavy industrial areas on urban trees and to ensure successful and effective afforestation in the vicinity of mega cities in China, further investigations of the plant metabolism are urgently needed and to be conducted over the long-term (at least three years) on adult trees and under extreme environmental conditions as they occur in heavy industrial areas in China.

Acknowledgements： The research work was supported by the National Natural Science Foundation of China (No. 41675153, 31870458, 31270518, 31370601, 31170573 and 41501227) and Sichuan Science and Technology Program (2018JY0086). We appreciate Dali Tao for critical review of the manuscript, Ms. Donglan Xiong and Dr. Qin Ping for their help in this study.

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Table 1. Growth parameters of tissues in Catalpa ovata seedlings exposed to elevated soil Cd and/or O3 concentrations. Treatments

Leaf (g)

Stem (g)

Root (g)

Total biomass (g)

R/S ratio

AA Cd-0

10.13±0.84a

31.55±4.93a

27.83±4.85a

71.88±8.53a

0.63±0.06a

Cd-100

6.45±2.57b

22.85±3.34b

15.01±2.99b

46.51±5.46bc

0.47±0.06a

Cd-500

5.26±1.32b

21.59±3.84b

14.87±2.31b

43.18±3.93bc

0.54±0.16a

Cd-0

10.21±0.91a

27.02±1.01ab

22.72±4.87a

61.50±6.76a

0.58±0.10a

EO

Cd-100

9.24±1.58ab

21.53±1.96b

16.22±4.97b

47.98±7.16b

0.51±0.12a

Cd-500

6.17±2.41b

17.87±2.60b

11.02±1.25b

35.63±6.52c

0.45±0.05a

O3

ns

ns

ns

ns

ns

Cd

**

**

***

***

ns

O3 × Cd

ns

ns

ns

ns

ns

Data are shown as mean ± standard deviation (SD) (n=3). O3 treatments are indicated as ambient air (AA; control; 40 µg O3/m3) and elevated O3 (EO; 120 µg O3/m3). Cd soil treatments are indicated as (Cd-0) 0 mg Cd/kg, (Cd-100) 100 mg Cd/kg, and (Cd-500) 500 mg Cd/kg. R/S (root/shoot), shoot means leaf plus stem. Different letters in the same column represent statistically significant differences between treatments (P < 0.05). General liner model (GLM) was applied to examine the individual and interactive effects of Cd and O3 on different parameters. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: P > 0.05.

Figure legends: Fig. 1. Physiological parameters in leaves of Catalpa ovata seedlings exposed to elevated soil Cd and/or O3 concentrations. A-D: net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr). E-H: malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and abscicic acid (ABA) content. O3 treatments are indicated as ambient air (AA; control; 40 µg O3/m3) and elevated O3 (EO; 120 µg O3/m3). Cd soil treatments are indicated as (Cd-0) 0 mg Cd/kg, (Cd-100) 100 mg Cd/kg, and (Cd-500) 500 mg Cd/kg. Different letters represent statistically significant differences between treatments (P < 0.05). General liner model (GLM) was applied to examine the individual and interactive effects of Cd and O3 on different parameters. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: P > 0.05.

Fig. 2. Changes in Cd, Mg, Fe and Zn content in different plant tissues (leaf, stem and root) of Catalpa ovata seedlings exposed to elevated soil Cd and/or O3 concentrations. O3 treatments are indicated as ambient air (AA; control; 40 µg O3/m3) and elevated O3 (EO; 120 µg O3/m3). Cd soil treatments are indicated as (Cd-0) 0 mg Cd/kg, (Cd-100) 100 mg Cd/kg, and (Cd-500) 500 mg Cd/kg. Different letters represent statistically significant differences between treatments (P < 0.05). General liner model (GLM) was applied to examine the individual and interactive effects of Cd and O3 on different parameters. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns: P > 0.05.

Fig. 1.

a

a a

a

a

8 b

c

b

5

gs (mmol/m2 · sec)

4

c

(E)

0 1.2

(B)

12

b

10

1

16

b

15

(A)

a

Cd: ***; O3: ns; Cd×O3: ns

Cd: ***; O3: ns; Cd×O3: ns

Cd: ***; O3: ns; Cd×O3: ns

a

a

a

0.8

a

a Cd: ns; O3: ns; Cd×O3: ns a a a a

a

500

b

250

(F)

0 0.3

Cd: ***; O3: **; Cd×O3: ns

a

a

b

bc

Ci (µmol/mol)

b

Cd: **; O3: **; Cd×O3: ns

b

c

ab

0.25 0.2

ab 0.15 bc c

0.1 c 0.05 0 200

b

b a

9

a

c

d

a e

d

b

150 100

f

6 c

50

3

0

0 AA

AA

EO

EO Treatments

Treatments

Fig. 2. 600 Leaf

Stem

Cd content (mg/kg)

500

Root

Cd: **; O3: ns; Cd×O3: ns a

a

400

a

a

300 200

(G)

Cd: ***; O3: ***; Cd×O3: ***

Cd: ***; O3: **; Cd×O3: ns Tr (µmol/m2 · sec)

1500

750

b

0.2

(D)

1750

1000

0.4

12

0 2000

1250

0.6

0 500 450 400 350 300 250 200 150 100 (C) 50 0 15

MDA content (µg/g FW)

20

Cd-500

SOD activity (U/g FW)

20

Cd-100

POD activity (U/g FW)

Pn (µmol/m2 · sec)

25

Cd-0

b b

ABA content (ng/g FW)

30

(H)

Graphical abstract

Elevated O3 OTCs simulation Heavy industrial areas

 Cd but not O3 negatively affected

Highlights 1. Soil Cd addition significantly reduced the growth of Catalpa ovata seedlings 2. Elevated O3 exposure did not affect growth, photosynthetic rate and oxidative injury of C. ovata seedlings 3. No interactive effect between Cd and O3 on physiological processes of C. ovata was found 4. C. ovata is more tolerant to elevated O3 exposure than to Cd soil contamination

Conflicts of interest The authors declare that they have no conflict of interest.