Effects of elevated ozone on physiological, anatomical and ultrastructural characteristics of four common urban tree species in China

Document downloaded from: This paper must be cited as: The final publication is available at Copyright Additional Information https://dx.doi.org/10.1016/j.ecolind.2016.03.012 http://hdl.handle.net/10251/77345 Elsevier Gao, F.; Calatayud Lorente, V.; García-Breijo, F.; Reig Armiñana, J.; Feng, Z. (2016). Effects of elevated ozone on physiological, anatomical and ultrastructural characteristics of four common urban tree species in China. Ecological Indicators. 67:367-379. doi:10.1016/j.ecolind.2016.03.012. 1 Effects of elevated ozone on physiological, anatomical and ultrastructural 1 characteristics of four common urban tree species in China 2 3 Feng Gaoa,1, Vicent Calatayuda,b,1, Francisco García-Breijoc,d, José Reig-Armiñanac, 4 Zhaozhong Fenga* 5 6 aState Key Laboratory of Urban and Regional Ecology, Research Center for 7 Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, 8 Haidian District, Beijing 100085, China 9 bFundación CEAM, c/Charles R. Darwin 14, Parque Tecnológico, 46980 Paterna, 10 Valencia, Spain 11 cLaboratorio de Anatomía e Histología Vegetal ‘‘Julio Iranzo’’, Jardín Botánico, 12 Universitat de València, c/Quart, 80, 46008 Valencia, Spain. 13 dDepartamento de Ecosistemas Agroforestales. ETSIAMN. Universidad Politécnica 14 de Valencia. Camino de Vera s/n, 46022-Valencia, Spain. 15 16 17 *Corresponding author: Zhaozhong Feng. E-mail: [email protected]n, Tel: +86-1018 62943823, Fax: +86-10-62943822; 19 1Both authors contributed equally to this work. 20 *Manuscript Click here to view linked References 2 Abstract 21 22 Fast urbanization has led to ozone (O3) being the main pollutant in summer in most of 23 China. To assess future ground-level O3 effects on the service of urban greening 24 species and clarify the underlying mechanism of O3 damage, four common urban 25 greening species of Ailanthus altissima (AA), Fraxinus chinensis (FC), Platanus 26 orientalis (PO) and Robinia pseudoacacia (RP) were exposed to non-filtered air (NF) 27 and to elevated O3 (E-O3) in open-top chambers. E-O3 induced visible injury in all 28 species as well as microscopic alterations such as collapse of the palisade parenchyma 29 cells, callose accumulation, or chloroplast and mitochondrial accelerated senescence. 30 E-O3 significantly reduced light-saturated CO2 assimilation (Asat), the maximum 31 activity of Rubisco (Vcmax), the maximum electron transport rate (Jmax), and 32 fluorescence parameters such as the quantum yield of noncyclic electron transport 33 (ɸPSII), and the quenching of photochemical efficiency of PSII (qP). It also increased 34 total antioxidant capacity, phenolics and ascorbate contents. No significant interaction 35 between O3 and species was found in photosynthetic performance and antioxidant 36 systems, suggesting that the four species selected were sensitive to O3. Of all four 37 species, AA was the most sensitive species due to a combination of earlier injury 38 onset, anatomical features, lower antioxidant and higher stomatal conductance. The 39 sensitivity of tree species to O3 is a factor to be considered for urban greening. Ozone 40 may affect important urban forest ecosystem services by reducing CO2 assimilation. 41 42 Keywords 43 Antioxidant system, Ozone, Photosynthesis, Ultrastructure, Urban greening species 44 45 Capsule 46 Ground-level ozone negatively affects common greening tree species in China 47 3 Abbreviations 48 A = photosynthetic rate 49 AA = Ailanthus altissima 50 AOT40 = accumulated hourly O3 concentration over a threshold of 40 ppb during 51 daytime 52 AsA = ascorbate 53 Asat = light-saturated photosynthesis 54 BVOC = biogenic volatile organic compounds 55 Ca = ambient CO2 concentration 56 Car = carotenoid 57 Chl = chlorophyll 58 Ci = intercellular CO2 concentration 59 Fv’/Fm’ = actual photochemical efficiency of PSII in the saturated light 60 FC = Fraxinus chinensis 61 ɸPSII = the quantum yield of noncyclic electron transport. 62 gs = stomatal conductance 63 Jmax = the maximum rate of electron transport 64 LM = light microscopy 65 LMA = leaf mass per area 66 Ls = stomatal limitation to photosynthesis 67 O3 = ozone 68 OTC = open-top chambers 69 PBS = phosphate buffered saline solution 70 PO = Platanus orientalis 71 RP = Robinia pseudoacacia 72 TEM = Transmission Electron Microscopy 73 qP = quenching of photochemical efficiency of PSII 74 Vcmax = the maximum carboxylation efficiency 75 WUE = water use efficiency 76 77 4 1. Introduction 78 Tropospheric ozone (O3) levels are of great concern as this pollutant affects human 79 health, ecosystem services and food security besides being a greenhouse gas (The 80 Royal Society, 2008; IPCC, 2013). For sensitive plants, high O3 concentration is 81 known to induce visible injury, impair photosynthesis, produce reductions in growth 82 and yield, and alter plant interactions with pests and diseases (Krupa et al., 2000). 83 Projected changes of the annual daily mean maximum eight-hour (DM8H) 84 surface O3 concentrations are expected to be in the range of 2 to 8 ppb, -3 to 8 ppb, 85 and -7 to 9 ppb for the 2020s, the 2050s, and the 2090s in summertime for the whole 86 East Asia (Lee et al., 2015). Ozone concentrations in China are rising at a higher rate 87 than in other countries because O3 precursors (mainly NO2) have steadily increased at 88 annual growth rate of 5% caused by its fast industrialization and urbanization (Wang 89 and Mauzerall, 2004; Feng et al., 2015a). In Beijing, the monthly average of peak O3 90 concentrations is currently 100 ppb in July, while the AOT40 (accumulated hourly O3 91 concentration over a threshold of 40 ppb during daytime) from June to August is 29 92 ppm.h (Feng et al., 2015b; Yuan et al., 2015). The yearly average of O3 concentration 93 in Beijing cities reaches more than 60 ppb during May 2014 to April 2015 (Chen et al., 94 2015), and short-term projected emissions suggest that O3 concentration will further 95 increase (Yamaji et al. 2008). Therefore, current O3 levels are, and future O3 levels are 96 expected to be by far above the threshold value of 40 ppb and AOT40 critical level of 97 5 ppm.h, which have been established to protect sensitive plants against O3 (LRTAP, 98 2010). 99 Cities are characterized by higher levels of pollutant emissions, energy 100 consumption and higher temperatures (heat island effect) than surrounding areas. One 101 of the multiple environmental benefits of vegetation is the improvement in air quality 102 (Nowak et al., 2014). In the central part of Beijing, the removal of pollutants by trees 103 was quantified to be 1261.4 tons in 2002, mostly particles (61%), with O3 accounting 104 for 20% (Yang et al., 2005). However, biogenic volatile organic compounds (BVOCs) 105 emitted from vegetation such as isoprene and monoterpenes are precursors of O3 (The 106 5 Royal Society, 2008), so the contribution of urban trees to O3 formation can even 107 offset their removal capacity (Yang et al., 2005). Therefore, it is important to select 108 plants with low BVOC emission rates, high pollutant removal capacity and also 109 tolerant to air pollutants when planting trees in cities. The present paper focuses on 110 the latter aspect. 111 In Beijing, O3 concentrations are high enough to induce visible injury in 112 sensitive species and cultivars, including several ornamental trees (Feng et al., 2014). 113 These symptoms are observed in large gardens or urban forests in parks inside the city, 114 or in crop areas or tree plantation surrounding the city rather than in streets where 115 high NO traffic emissions locally scavenge O3 (due to the titration effect). Ozone 116 effect on plants depends both on the O3 dose entering the plant through the stomata 117 which is directly related to water vapor stomatal conductance (gs), and also on their 118 defense ability to cope with oxidative stress (Matyssek et al., 2007; Paoletti et al., 119 2008). On the other hand, leaf functional traits are considered to play a role in O3 120 sensitivity, e.g. plants with higher leaf mass per area (LMA), or higher thickness or 121 density of mesophyll tissues being tolerant to O3 (Bussotti, 2008; Zhang et al., 2012). 122 However, information on the effects of current and predicted future O3 levels on 123 urban greening species is still very scarce in China. In the present study, we exposed 124 four commonly planted urban greening tree species to elevated O3 level which is the 125 representative of a future scenario by 2050 on the basis of annual increase rate of 0.73 126 ppb/year at Shangdianzi station observation nearby Beijing city (Dr. XB Xu, personal 127 communication) and an increase rate of 0.5-2% at a global scale (Vingarzan, 2004). 128 The four species are the tree of heaven (Ailanthus altissima (Mill.) Swingle, AA), the 129 Chinese ash (Fraxinus chinensis Roxb., FC), the American sycamore (Platanus 130 orientalis L., PO) and the black locust (Robinia pseudoacacia L., RP) . Three of them 131 (AA, FC, RP) are regarded as O3 sensitive. This study tests the following two 132 hypotheses: (1) O3 sensitivity differs among investigated species, considering 133 anatomical and ultrastructural changes, photosynthetic performance and antioxidant 134 6 systems; (2) plants with a higher stomatal conductance, lower antioxidant capacity 135 and thinner leaves are more sensitive to O3. 136 137 2. Materials and methods 138 2.1. Plant materials 139 One-year-old seedlings of AA, FC, PO and RP were obtained from a commercial 140 nursery near the experimental site. Bare rooted seedlings were planted in 20 L circular 141 plastic pots on 31 March 2013 and grown at ambient field condition. Pots were filled 142 with native light loamy soil (pH 7.96, Organic C 14.7 g/kg; total N 1.64 g/kg, 143 available P 6.59 mg/kg, available K 139.8 mg/kg) randomly selected from a nearby 144 farmland. Plants with similar height and basal diameter were selected. Ten days 145 before O3 fumigation, they were pre-adapted to open-top chamber (OTC, octagonal 146 base, 12.5 m2 of growth space with a diameter of 4 m, and 3.0 m in height). All plants 147 were watered at field capacity at 1-3 day intervals to avoid water stress. Solid, 148 slow-release fertilizer (N/P/K = 17,17,17) was applied at a rate of 300 kg ha-1 to each 149 plant at July during the experiment. 150 151 2.2. O3 treatments 152 The experiment was carried out at Changping (40°19′N, 116°13′E), Northwest 153 Beijing. The area has a semi-humid continental climate, with a yearly precipitation of 154 550 mm, and an annual mean temperature of 11.8 °C. Plants were exposed to two O3 155 treatments in OTCs for four and a half months (from 1 June to 15 October): 156 non-filtered ambient air (NF, averaged O3 concentration of 42 ppb from 09:00 to 157 18:00), and NF supplied with 40 ppb of O3 (E-O3, averaged O3 concentration of 69 158 ppb from 09:00 to 18:00). The four species and two O3 treatments were selected for 159 the present study from a wider investigation involving a total of 10 species and six 160 different O3 regimes in six OTCs. Positional effects were avoided by changing plant 161 positions within each OTC weekly, and by switching them randomly among six OTCs 162 monthly (Feng et al., 2011a). For each O3 treatment, 4-6 plant replicates were used for 163 7 each species. Ozone was generated from pure oxygen using an O3 generator (HY003, 164 Chuangcheng Co., Jinan, China), mixed with ambient air and then piped into OTCs 165 through a PVC tube (11 cm in diameter) using a fan (1.1 kW, 1080 Pa, 19 m3 min-1, 166 CZR, Fengda, China). The flow rate of pure oxygen was regulated by mass flow 167 controllers so as to achieve the target O3 concentration at the top of the canopy in the 168 fumigation treatments. An O3 analyzer (Model 49i-Thermo, USA) was used to 169 continuously monitor O3 concentrations inside the OTCs via a Teflon solenoid valve 170 switch system connected to a set of Teflon tubes (4 mm in diameter), which collected 171 air from sampling points at approximately 10 cm above the plant canopy in each 172 chamber. The monitors were calibrated by a 49i-PS calibrator (Thermo Scientific, 173 USA) before the experiment and once a month during the experiment. The daily 174 maximum fumigation period was 9 h (from 09:00 to 18:00) through a fan running 175 when there was no rain, fog, mist, or dew, according to the protocols in free air O3 176 concentration enrichment system (Feng et al., 2011b). The monthly ambient O3 177 concentration (from 09:00 to 18:00) in the open field was ranged from 43 ppb 178 (September) to 69 ppb (June), with the highest one hour peak being 153 ppb (at 16 h 179 on 20 September) . 180 181 2.3. Visible injury 182 Visible injury was assessed weekly in all plants (4-6 per O3 treatment). The 183 percentage of injured leaves (for PO, with simple leaves) or leaflets (for the rest of 184 species, with composite leaves) per plant was scored, in order to classify each plant 185 according to the following classes: 0, no leaves injured; 1, ≤1% leaves injured; 2, >1% 186 – ≤10% leaves injured; 3, >10% – ≤50% leaves injured; 4, >50% leaves injured. 187 188 2.4. Leaf traits 189 Twenty-five asymptomatic mature leaves from NF plants were collected studying the 190 leaf traits using a scanner and ImageJ software (Gao et al., 2011). LMA was 191 calculated as dry mass (mg) / leaf surface area (cm2). Leaf dry mass was determined 192 8 by oven-drying leaves at 60 °C for 40 h until steady weight. To estimate the leaf traits, 193 paraffin-embedded sections of six leaves or leaflets (five sections per leaf or leaflet, 194 and five measurements made per section) were examined under the microscope 195 following the methods described below. 196 197 2.5. Microscopy examinations 198 To study the effects of O3, six symptomatic and six asymptomatic leaves or leaflets 199 from the upper canopy layer (6-8th fully expanded leaves from the top) of three plants 200 for each species were collected in August from E-O3 and NF plants, respectively, 201 before the onset of senescence. Samples were fixed in 2% Karnovsky fixative for 8 h 202 at 4°C, then washed three times for 15 min with 0.01 M PBS (pH 7.4). For 203 microscopy examinations, leaf portions were submitted to freeze-cut, paraffinand 204 resin-embedded sections. For freeze-cut sections (~30 µm), a freezing microtome 205 (CM 1325; Leica, Germany) was used, paraffin-embedded sections (~10 µm) were 206 cut with an Anglia Scientific microtome, and Spurr’s resin-embedded samplers were 207 cut with a diamond knife (DIATOME Histo 45°) and an ultramicrotome (Ultratome 208 Nova LKB Bromma) (~1.5 µm). Freeze-cut sections were observed under 209 epifluorescence BV (autofluorescence) or stained with aniline blue and observed 210 under epifluorescence UV to detect callose depositions. For fluorescence microscopy, 211 an Olympus U-ULS 100 HG epifluorescence system with U-MWU (excitation filter 212 330–385 nm, dichroic mirror 400 nm, barrier filter 420 nm) and U-MWBV (excitation 213 filter 400–440 nm, dichroic mirror 455 nm, barrier filter 475 nm) cubes was used. 214 Paraffin-embedded sections were stained with safranin-fast green, or with trichromic 215 FSA for observation of the different structures and for identifying their composition. 216 Semi-thin sections were stained with toluidine blue. Pectinaceous drops were detected 217 with this stain. All light microscope (LM) observations were carried out by an 218 Olympus Provis AX 70 fluorescence microscope equipped with an Infinity 2-3C 219 Lumenera® digital camera and analyzed with “Infinity Analyze” Software v.6.4.1. 220 For transmission electron microscopy (TEM), samples were fixed as LM, then 221 15 340 3.3 Anatomical and ultrastructural changes induced by E-O3 341 LM and TEM studies showed the effects of O3 as well as plant responses against 342 oxidative stress. Palisade parenchyma cells were in general the most affected part of 343 the mesophyll (AA > PO > FC > RP; see Fig. 3). The middle lamella of the cell wall 344 was degraded (Figs 4A, 5B) and small pectinaceous drops produced by its 345 degradation were observed (Figs. 4A, 5A, 5E, 5F, 6A). Cell walls were progressively 346 altered (Figs. 3C,3F, 4B, 4C, 5B, 5C, 7C, 7E), and fluorescence LM (Fig. 3A) and 347 TEM (Figs. 5A, 5B, 6B) showed that callose was accumulated between the membrane 348 and cell wall, especially in AA and PO. Inside the cells, vacuole content becomes 349 altered. In AA and RP, vacuolar content of the affected cells became denser by the 350 accumulation of tannins (Figs. 3B, 3H), and in PO, the normal cells of which have 351 vacuoles with abundant tannic content, a gradual coagulation of tannins in the 352 affected cells was observed (Figs. 5E, 5F), as well as a large accumulations of crystals 353 in their cytoplasm (Figs. 5D, 5D, 6C). Finally, tonoplast broke and cells lost turgor 354 (Fig. 7B). In a later stage, cells collapsed (Figs. 3, 4A, 4C, 5B, 5E, 5F, 7E) leading to 355 an increase of intercellular spaces (AA, PO > FC > RP). Chloroplasts were also 356 strongly affected in all species, increasing electrodense material and changing their 357 shape. Accumulation of big starch grains (Figs. 4A, 5B, 5C, 7A), abundant 358 plastoglobuli (Figs. 4A, 7A, 7B, 7C, 7E), and lipid-protein bodies (especially in AA, 359 Figs. 4A, 4B, 4D) were also observed. Finally, thylakoid membranes were partly or 360 totally disaggregated (Fig. 7B). Mitochondria also experienced degradation processes. 361 In some cases, the accumulation of lipid droplets similar to plastoglobuli was 362 observed, especially in AA (Fig. 4D). Similar but much less conspicuous changes 363 were observed in the spongy parenchyma. Upper and lower epidermises were not 364 distinctly affected in any of the species with an exception of AA and RP, in which 365 some epidermal cells can collapse (Figs. 3A, 3G, 4C). In some cases, chloroplast of 366 the guard cells of stomata were also affected, as indicated by large starch 367 accumulations (Fig. 7A). In the vascular bundles, xylem was never affected but 368 16 phloem cells can experience changes in shape and callose deposition may increase in 369 the sieve tubes (Figs. 3H, 6E, 7D). 370 371 Figure 3. (A) Fluorescence micrograph (UV) of a cross section of Ailanthus 372 17 altissima leaf stained with aniline blue showing an affected area (asterisks) with 373 numerous collapsed cells and with callose depositions (CaD) on its cell walls. (B) 374 Micrograph of a cross section of an Ailanthus altissima affected leaf stained with 375 safranin-fast green showing numerous collapsed cells (asterisks) with tannic (Ta) 376 contents inside. (C) Micrograph of a cross section of a Fraxinus chinesis affected leaf 377 stained with trichromic FSA showing numerous collapsed cells (asterisks) with tannic 378 (Ta) contents inside. (D) Autofluorescence micrograph (BV) of a cross section of 379 Fraxinus chinensis leaf showing an affected area (asterisks) of palisade parenchyma 380 (PP) without chlorophylls. (E) Micrograph of a semithin cross section of a Platanus 381 orientalis affected leaf stained with toluidine blue. Numerous collapsed cells 382 (asterisks) leaving between them large intercellular spaces (IS) are observed. (F) 383 Micrograph of a cross section of a Platanus orientalis affected leaf stained with 384 trichromic FSA showing collapsed cells (asterisks) with affected cell wall (ACW) 385 and tannic (Ta) contents inside. (G) Micrograph of a semithin cross section of a 386 Robinia pseudoacacia affected leaf stained with toluidine blue. A collapsed epidermis 387 (CEp) is observed. (H) Autofluorescence micrograph (BV) of a cross section of a 388 Robinia pseudoacacia affected leaf. Numerous cells filled with tannin content (Ta) 389 are observed. Others abbreviations. AbEp: abaxial epidermis; AdEp: Adaxial 390 epidermis; Cu: cuticle; TVB: tertiary vascular bundle; SP: spongy parenchyma; St: 391 stomata; SVB: secondary vascular bundle. 392 393 18 394 Figure 4. Details of TEM micrographs of cross sections of Ailanthus altissima leaves. 395 (A) Two collapsed cells (CC) of palisade parenchyma with great intercellular spaces 396 (IS). Pectinacious drops (PD) and degraded middle lamella (DML) are observed. 397 (B) Cells of palisade parenchyma with abundant gerontoplasts (Gp) filled with 398 abundant lipid-protein bodies (LPB), degraded cell wall (DCW) and great 399 intercellular spaces (IS). The area marked with a white frame is shown magnified at 400 19 the bottom right of the image. (C) Collapsed cells (CC) of palisade parenchyma and 401 adaxial epidermis (EpCC) showing a degraded cell wall (DCW). (D) Detail of a cell 402 with several mitochondria (Mi) containing some mitoglobuli (Mg). Other 403 abbreviations. AdEp: adaxial epidermis; N: nucleus; Pd: plasmodesma; Pg: 404 plastoglobuli; St: starch; ThM: thylakoidal membranes; Va: Vacuole. 405 406 20 Figure 5. A to B: Details of TEM micrographs of cross sections of Fraxinus chinensis 407 leaves. D to F: Details of TEM micrographs of cross sections of Platanus orientalis 408 leaves. (A) Spongy parenchyma cell showing an altered medium lamella. Some 409 pertinacious drops (PD) and callose depositions (CaD) between the cell membrane 410 and cell wall are observed. (B) Cells of spongy and palisade parenchyma showing 411 different alterations. Some of them are collapsed (CC). (C) Detail of palisade 412 parenchyma cells. Degraded cell wall (DCW) and gerontoplasts (Gp) are observed. 413 (D) Cells showing a crystalized cytoplasm (CrCy). In (D1) a detail of this 414 crystallization is observed. (E) Affected palisade parenchyma cells. Collapsed cells 415 (CC), vacuoles with tannins (Ta), crystalized cytoplasm (CrCy) and numerous 416 chloroplasts (Chl) with plastoglobuli (Pg) are observed. (F) Cells of palisade 417 parenchyma showing varying degrees of decomposition of tannins (Ta) within the 418 vacuoles (Va). Other abbreviations. AChl: Altered chloroplast; AdEp: adaxial 419 epidermis; Cr: (calcium oxalate) crystal; DML: degraded medium lamella; N: 420 nucleus; S: starch; Xy: xylem. 421 422 423 21 424 Figure 6. (A). TEM micrograph of cross sections of Platanus orientalis leaf. Detail of 425 spongy parenchyma cells showing pertinacious drops (PD). (B). TEM micrograph of 426 cross sections of Platanus orientalis leaf. Callose depositions (CaD) between the cell 427 membrane and cell wall are observed. (C). TEM micrograph of cross sections of 428 Platanus orientalis leaf. Cell showing a crystalized cytoplasm (CrCy). (D). 429 Micrograph of a cross section of a Fraxinus chinesis affected leaf (left) and a control 430 leaf (right) stained with toluidine blue. The affected leaf shows a collapsed phloem 431 (CoPh). Other abbreviations. AChl: altered chloroplast; Cr: crystals; CrCy: 432 crystalized cytoplasm; DCW: degraded cell wall; Gp: gerontoplast; LPB: 433 lipid-protein body; Mi: mitochondria; N: nucleus; Ph: Phloem; S: starch Va: Vacuole; 434 Xy: xylem. 435 22 436 Figure 7. Details of TEM micrographs of cross sections of Robinia pseudoacacia 437 leaves. (A) Detail of a stoma (ST) with affected chloroplast and spongy parenchyma. 438 Chloroplast (Chl) in cells of spongy parenchyma are affected and contain numerous 439 plastoglobuli (Pg). The area marked with the dashed square is shown in Figure (B). (B) 440 Detail of affected chloroplasts containing numerous plastoglobuli and many grana 441 disorganized (AGr). The tonoplast (To) is disintegrated into many areas (DTo). (C) 442 23 Palisade parenchyma cells containing chloroplasts with numerous plastoglobuli and 443 degraded cell walls (DCW) in some areas. (D) Detail of a tertiary vascular bundle 444 with the affected phloem. Callose depositions (asterisks) are observed in some 445 phloem (Ph) vessels. (E) Cells affected in the spongy parenchyma. Their cell walls 446 are degraded (DCW) and contain chloroplasts with numerous plastoglobuli (Pg). 447 Other abbreviations. AbEp: abaxial epidermis; AdEp: Adaxial epidermis; BS: Bundle 448 sheath; Gr: grana; Mi: mitochondria; N: nucleus; OC: occlusive cells; S: starch; Ta: 449 tannins; Va: vacuole; Xy: xylem. 450 451 3.4. Leaf gas exchange and chlorophyll a fluorescence 452 Effects of E-O3 on gas exchange and fluorescence parameters were significant for Asat, 453 ɸPSII, and qP (Table 2). There were no significant interactions between species and O3, 454 suggesting the responses to E-O3 were the same among all species (Table 2). Effects 455 on Asat increased with O3 exposure, as indicated that there was a significant reduction 456 in Asat by 27%, 21%, 17%, and 31% for AA, FC, PO and RP, respectively, in 457 September (Tables 2 and 3). For the same month ɸPSII was significantly reduced by 13% 458 and 21% in PO and RP, respectively (Table 3). Ci and WUE were not significantly 459 affected by O3, while gs was significantly reduced in AA and RP in September. 460 Notably, there was significant difference among species in gs (Table 2), as shown by 461 highest gs values in AA (0.15 mol m-2 s-1) and lowest in RP (0.07 mol m-2 s-1), with FC 462 and PO showing intermediate values (0.13 mol m-2 s-1). 463 24 Table 2. Analysis of variance of the effects of O3, species and sampling date, and their 464 interactions on gas exchange and chlorophyll a fluorescence parameters, pigment and 465 antioxidant contents. 466 467 O3 Species O3×Species Date O3×Date O3×Date ×Species Asat 0.0002 <0.0001 0.9983 0.2049 0.0096 0.8472 gs 0.5411 0.0002 0.5788 0.8176 0.0096 0.2809 Ci 0.7889 <0.0001 0.2548 0.7265 0.3888 0.3006 WUE 0.9437 0.1959 0.5503 <0.0001 0.3258 0.3105 Fv'/Fm' 0.6297 0.0002 0.0773 0.1689 0.3076 0.8374 ɸPSII 0.0427 <0.0001 0.5481 <0.0001 0.4844 0.7984 qP 0.0033 0.0065 0.6313 <0.0001 0.7380 0.3661 Vcmax <0.0001 0.4209 0.5318 0.0337 0.8005 0.2668 Jmax <0.0001 0.0896 0.3419 0.1311 0.9928 0.3479 Ls 0.0006 0.2542 0.4295 0.2473 0.0148 0.9018 TAC <0.0001 <0.0001 0.0965 Phenolics <0.0001 <0.0001 0.7386 Total AsA <0.0001 <0.0001 0.0908 Reduced AsA <0.0001 <0.0001 0.0799 Chl a 0.0825 0.0991 0.8684 Chl b 0.081 0.0403 0.9558 Chl a+b 0.0815 0.0822 0.891 Car 0.0715 0.0183 0.7867 Chla/Chlb 0.2442 0.0200 0.816 468 31 et al., 2008; Calatayud et al., 2011). Phenolic metabolites are suggested to play a 552 protective role against oxidative stress as antioxidants (Kangasjärvi et al., 1994). In 553 the four species, E-O3 increased accumulation of phenolic compounds. In the 554 vacuoles of AA and PO, rich in condensed tannins, these compounds changed their 555 even distribution by a coagulated aspect most likely due to oxidative processes 556 (Vollenweider et al., 2003). Changes in vacuole tannins due to O3 have been observed 557 in the mastic plant (Reig-Armiñana et al. 2004). In RP, less rich in tannins, an 558 accumulation of phenolic compounds is observed. Previous microscopy studies have 559 shown that accumulation of phenolic compounds including tannins and anthocyanins 560 can be induced by O3 in some species (Vollenweider et al., 2003; Bussotti et al., 561 2005). 562 At the ultrastructural level, changes in organelles that may be considered as an 563 acceleration of the natural foliar senescence process can be observed. Foliar 564 senescence is characterized by a decline in whole leaf gas exchange and protein levels, 565 leaf yellowing, and the chloroplast-to-gerontoplast transition. Ultrastructurally, 566 gerontoplast development is seen primarily as a progressive unstacking of grana, a 567 loss of thylakoid membranes and a massive accumulation of plastoglobuli and other 568 lipid-protein inclusions (Harris & Schaefer, 1981). In our study, ultrastructural 569 changes in the chloroplast included an increase in electrodense material, accumulation 570 of starch, plastoglobuli and lipid-protein bodies, thylakoid degradation, membrane 571 disruption and changes in shape. Starch accumulation may be related to a difficulty in 572 sucrose transport outside the chloroplasts and to other tissues (Calatayud et al., 2011). 573 Landolt et al. (1997) found enhanced content of soluble sugars in leaves of 574 O3-exposed birch plants, and in the same species, Matyssek et al. (1992) observed 575 accumulation of starch along veins, which suggested a reduction of carbon export 576 from source leaves. This can be related with altered cell membranes and impaired 577 phloem loading induced by O3 (Grantz, 2003). In O3-injured leaves, phloem cells can 578 experience evident changes in shape, which may obviously impair their functionality 579 and affect sucrose translocation (Calatayud et al., 2011). On the other hand, an 580 32 increase in plastoglobuli can originate from the lipid-soluble degradation products 581 from the thylakoid membranes (Matile, 1992; Kivimäenpäa et al., 2010). Overall, the 582 four species show partly similar anatomical and ultrastructural changes that are 583 related both to O3 damage to different cell components, defence responses and 584 accelerated senescence. Notably, changes were more evident in AA, with many 585 palisade parenchyma cells collapsed leading to a large intercellular spaces, distinct 586 callose dipositions, denser vacuolar content due to tannin accumulation, and more 587 altered chloroplasts (with accumulation of abundant lipid-protein bodies). On the 588 contrary, much less evident changes were observed in RP, with FC and PO being 589 intermediate. The damage to chloroplast functionality has led to reduced 590 photosynthesis (Table 3). Investment in defence may, on the other hand, reduce 591 carbon availability for plant growth. 592 Compared to control, changes in leaf gas exchange, chlorophyll a fluorescence 593 and carboxylation parameters by E-O3 showed similar responses in all species, as 594 indicated by no significant interactions between O3 and species. Ozone induced 595 significant declines in Asat and in chlorophyll a fluorescence parameters (ɸPSII and qP). 596 This decline in photosynthetic CO2 assimilation was associated with significant 597 reductions in Vcmax and Jmax, suggesting that biochemical limitations play an early and 598 primary role in the decline of CO2 assimilation by O3 (e.g., Calatayud et al., 2010; 599 Feng et al., 2011b; Cho et al., 2011). Changes in fluorescence parameters under 600 steady-state illumination may reflect a down-regulation process for adjusting the 601 production of reductive power and chemical energy to a lower demand by the 602 Calvin-Benson cycle (Calatayud et al., 2007). In a complementary study conducted 603 with the same plants but restricted to only three of the species (AA, FC and PO), E-O3 604 increased stomatal sluggishness (i.e. slowed stomatal response) with FC and PO being 605 the most and less affected species, respectively (Hoshika et al., 2014). Increased 606 sluggishness may be related to accelerated leaf senescence in the cell physiological 607 processes (Paoletti et al., 2009), although the underlying mechanisms are still under 608 investigation (Hoshika et al., 2014). In the present study, chloroplasts of the guard 609 33 cells are sometimes altered, which could partly impair the normal performance of 610 these cells. 611 E-O3 significantly increased total and reduced AsA contents in the leaves. 612 Activation of the AsA synthesis is a defence response against oxidative stress, as AsA 613 is a central metabolite in plant antioxidant system. It serves as a chemical scavenger 614 to protect plants by reducing free radicals, and also as a substrate of extracellular 615 enzymes such as the ascorbate peroxidase (APX) which detoxify peroxides; therefore 616 propagation of oxidative signaling diminishes (Burkey et al., 2006; Dizengremel et al., 617 2013). Besides, all species in the present study showed an increase in phenolics 618 contents. Similar results have been previously reported in other tree species (Oksanen 619 et al., 2013). Phenolic metabolites probably increase O3 tolerance of plants due to its 620 effectiveness as radicals and ROS scavengers (Grace, 2005; Langebartels et al., 2002). 621 Increased TAC levels are consistent with increased phenolics content as phenolic 622 compounds have been found to be the major contributors to the antioxidant properties 623 in extracts of different parts of 30 plants (e.g. Dudonné et al., 2009). In the present 624 study, however, all four species showed a similar response to O3 in antioxidant levels. 625 Besides the increases in AsA, phenols or TAC, the different species were not able to 626 counteract photosynthesis impairment, cellular and tissue damage, and visible injury, 627 suggesting that the possible contribution of these physiological responses was 628 insufficient to offset the high levels of oxidative stress. 629 Considering visible injury, anatomical and ultrastructural responses, AA is the 630 most sensitive of all species as it developed symptoms the earliest and the percentage 631 of injuried leaves was the highest from the beginning. Anatomical and ultrastructural 632 alterations were also the severest in this species. On the other side, any of the RP 633 plants reached the higher injury classes, and visible injury was much less conspicuous 634 than the other species, with anatomical and ultrastructural changes being also 635 moderate. FC and PO were intermediate, with some of the leaves strongly affected by 636 a marked yellow or brown stippling. Several studies have shown that LMA is well 637 correlated with O3 sensitivity of the species: plants with higher LMA are, in general, 638 34 more O3 tolerant, partly due to the fact that they have a higher chlorophyll and 639 nitrogen content per area unit, supporting a more efficient photosynthesis activity, 640 which can better feed detoxification processes (Bussotti, 2008; Zhang et al., 2012). 641 Our results are not in line with such a hypothesis, probably because the investigated 642 species have a narrow LMA range (5.0-9.1 mg cm-2). LMA is better related with O3 643 tolerance when a larger range among species is considered (e.g., comparing deciduous 644 with evergreen species, Calatayud et al., 2011). The higher sensitivity in AA can 645 rather be explained as a combination of factors including a thinner palisade 646 parenchyma layer, less active antioxidant responses to O3, and the highest gs. The 647 palisade parenchyma is the most photosynthetically active tissue, so a reduced 648 thickness may imply that less apoplast surface is available for defense reactions 649 (Dizengremel et al., 2013). The less active antioxidant responses in combination with 650 higher gs (related to a higher O3 uptake), would make this species more prone to 651 oxidative imbalances. These features are consistent with the pioneering, fast-growing 652 strategy of this species. On the other hand, although RP is also a sensitive species, its 653 lower gs and, probably also, its stronger antioxidant responses could better withstand 654 O3 stress, besides the fact of having lower constitutive antioxidant levels. Besides 655 showing more or less marked macroand microscopic responses, it is noteworthy that 656 photosynthesis was also impaired in all the species, with CO2 assimilation being 657 reduced. On the long run, such reductions are expected to affect CO2 fixation as plant 658 biomass, one of the services of urban forests. In the present study, however, only one 659 growing season was covered and the effects on biomass were negligible (data not 660 shown). 661 662 5. Conclusions 663 The investigated four species are sensitive to O3, as indicated by an increase in foliar 664 damage, accumulation of phenolic compounds in the leaf tissues, degradation of the 665 cell walls and organelles such as the chloroplasts, changes in antioxidant levels and 666 reduced photosynthesis rate. These changes might also alter plant interactions with 667 35 other abiotic and biotic stresses (Karnosky et al., 2005; Bussotti et al., 2008). The 668 current study suggests that sensitivity to O3 should be taken into account for urban 669 tree plantations in areas at risk of high ozone concentrations, avoiding very sensitive 670 species such as AA. This is especially relevant for urban forests and large tree 671 plantations in parks or in green belts around cities rather than for trees planted in 672 streets, where O3 is locally scavenged by reacting with NO traffic emissions. Further 673 studies of O3 exposure under controlled or semi-controlled conditions involving many 674 urban trees are still needed in order to support decision making for tree plantation in 675 ozone polluted cities of China. Other additional factors to be considered in urban 676 greening species in relation to air pollution are their rate of BVOC emission and 677 capacity to remove O3 and particulate matter (Paoletti et al., 2009; Calfapietra et al., 678 2013). 679 680 Acknowledgements 681 This study has been funded by the Hundred Talents Program, Chinese Academy of 682 Sciences and State Key Laboratory of Urban and Regional Ecology. Collaboration 683 between RCEES and Fundación CEAM has been possible thanks to project AMIS 684 (Fate and Impact of Atmospheric Pollutants, PIRSES-GA-2011-295132), and by the 685 Chinese Academy of Sciences Visiting Professorships for Senior International 686 Scientists (Grant Number: 2013T2Z0009). VC also acknowledges the support of 687 PROMETEOII/2014/038 project (Generalitat Valenciana, G.V.), and FGB and JRA 688 that of PROMETEOII2013/021 (G.V.), and CGL2012-40058-C02-01/02 (MINECO). 689 We thank Mr. Yulong Zhang for the experimental management. 690 691 6. 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