Environ Sci Pollut Res (2015) 22:2064–2075 DOI 10.1007/s11356-014-3481-8
RESEARCH ARTICLE
Effects of combined ozone and cadmium stresses on leaf traits in two poplar clones Antonella Castagna & Daniela Di Baccio & Anna Maria Ranieri & Luca Sebastiani & Roberto Tognetti
Received: 24 March 2014 / Accepted: 18 August 2014 / Published online: 30 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Information on plant responses to combined stresses such as ozone (O3) and cadmium (Cd) is scarce in tree species. On the other hand, high O3 concentrations in the atmosphere and heavy metal contaminations in water and soil simultaneously affect forest ecosystems. Toxic metals may exacerbate the consequences of air pollutants. In this research, two poplar clones, differently sensitive to O3 (“I-214” O3tolerant and “Eridano” O3-sensitive), were grown for 5 weeks in pots supplied with 0 and 150 mg Cd kg−1 soil and then exposed to a 15-day O3 fumigation (60 nl l−1, 5 h a day) or supplied with charcoal-filtered air under the same conditions (referred to as control samples). The effects of the two Responsible editor: Elena Maestri Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3481-8) contains supplementary material, which is available to authorized users. A. Castagna (*) : A. M. Ranieri Dipartimento di Scienze Agrarie, Alimentari e Agro-Ambientali, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy e-mail: [email protected] D. Di Baccio : L. Sebastiani BioLabs, Istituto di Scienze della Vita, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy R. Tognetti Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Contrada Fonte Lappone, 86090 Pesche, Italy R. Tognetti (*) The EFI Project Centre on Mountain Forests (MOUNTFOR), Via Edmund Mach 1, 38010 San Michele all’Adige, Italy e-mail: [email protected] Present Address: D. Di Baccio Istituto di Biologia Agroambientale e Forestale, Consiglio Nazionale delle Ricerche, Via Salaria km 29,300, 00015 Monterotondo Scalo, Italy
stressors, alone or in combination, on Cd accumulation, photosynthetic capacity, ethylene emission and oxidative state were investigated in fully expanded leaves. Cadmium accumulation in leaves caused a reduction, but not complete failure, of photosynthesis in Eridano and I-214 poplar clones. The reduction in assimilation rate was more important following O3 fumigation. Stomatal aperture after O3 treatment, instead, increased in I-214 and decreased in Eridano. Overall, Cd treatment was effective in decreasing ethylene emission, whereas O3 fumigation increased it in both clones, although interacting with the metal treatment. Again, O3 fumigation induced a significant increase in ascorbate (ASA) + dehydroascorbate (DHA) content, which was strongly oxidised by O3, thus decreasing the redox state. On the other hand, Cd treatment had a positive effect on ASA content and redox state in I-214, but not in Eridano. Although Cd and O3 are known to share some common toxicity pathways, the combined effects induced distinct clone-specific responses, underlying the complexity of plant reactions to multiple stresses. Keywords Ascorbic acid . Cd . Eridano . Ethylene . I-214 . O3 . Photosynthesis Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid Amax Net photosynthesis at saturating irradiance APX Ascorbate peroxidise ASA Ascorbate Cd Cadmium Ci Internal CO2 concentration DHA Dehydroascorbate Gw Stomatal conductance H2O2 Hydrogen peroxide NADPH Nicotinamide adenine dinucleotide phosphate NO Nitric oxide O3 Ozone
Environ Sci Pollut Res (2015) 22:2064–2075
[O3] PPFD ROS
O3 concentration Photosynthetic photon flux density Reactive oxygen species
Introduction Heavy metal pollution of soil and water has become a major worrying environmental issue worldwide, because of the contamination of large areas due to the anthropogenic activity (Pilon-Smits 2005). Among heavy metals, cadmium (Cd) is of particular concern due to its easy uptake by roots and high toxicity for plants (as well as animals). Cadmium can accumulate in roots up to micromolar concentration, before altering ultra-structural traits and browning root tips (Ederli et al. 2004). Being relatively mobile, part of the absorbed Cd can be translocated from roots and accumulated in shoots (Zacchini et al. 2011), causing chlorosis, inhibition of growth and even plant death, owing to its influence on stomatal behaviour, photosynthesis, respiration and water and nutrient uptake (e.g. Liu et al. 2011; Cocozza et al. 2014). Cadmium injury has been attributed to several factors, such as blocking of essential functional groups in biomolecules (Schützendübel et al. 2002) and displacement of metal ions from biomolecules (Rivetta et al. 1997). Cadmium may be involved in the suppression of antioxidant defence and in the overproduction of reactive oxygen species (ROS) (He et al. 2011; Iannone et al. 2010), which interfere with the redox status, possibly leading to uncontrolled oxidative stress (Rodríguez-Serrano et al. 2006). ROS increase could be indirectly caused by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in membranes (Romero-Puertas et al. 2004) and/or by reduction of activity/content of enzymatic and non-enzymatic antioxidants (Wójcik and Tukiendorf 2011). An open question is whether Cd accumulated in the foliage can have interactive effects with other factors of stress, eliciting ROS production. Tropospheric ozone (O3) is considered to be the most important air pollutant affecting vegetation (Ashmore 2005; Wittig et al. 2009). Prolonged exposures to relatively low O3 concentration [O3] (within the range of 20–50 nl l−1) affect plant health through stomatal uptake (Matyssek and Sandermann 2003; Panek 2004), causing leaf chlorosis (Ribas et al. 2005; Vollenweider et al. 2003), accelerated leaf senescence (Gielen et al. 2007; Zheng et al. 2002), reduced photosynthesis (Bagard et al. 2008; Di Baccio et al. 2008; Wittig et al. 2007) and decreased carbon allocation to sink tissues (Andersen 2003; Wittig et al. 2009), and affecting plant biomass and radial growth (Grulke et al. 2002; Panek and Goldstein 2001). In particular, leaf metabolism may be reoriented to repair processes and defence mechanisms (e.g. Cabané et al. 2004; Betz et al. 2009). Once O3 enters the leaf through stomata, it degrades rapidly in the apoplast to form various ROS (Langebartels et al. 2002; Vahala et al. 2003). Among ROS, the hydrogen
2065
peroxide (H2O2), in particular, is known to trigger a complex sequence of events leading to endogenous ROS production and activation of mitogen-activated protein kinase cascade, which in turn affects the synthesis of molecules such as ethylene, salicylic and jasmonic acids, involved in the signalling route and in modulation of the plant responses to the imposed stress (Kangasjärvi et al. 2005). However, when ROS production exceeds the plant ability to maintain the ROS concentration below a tolerance threshold, oxidative damage occurs (Castagna and Ranieri 2009). Among antioxidant metabolites, ascorbic acid, in addition to playing a key role in detoxification of H2O2 as a specific substrate for ascorbate peroxidase (APX), is capable of direct scavenging of O3 molecules and O3-derived ROS (Ranieri et al. 1999; Conklin and Barth 2004). Ethylene acts as a second messenger in oxidative signal transduction and regulates the induction and the spread of oxidative stress symptoms (Rao et al. 2002; Vahala et al. 2003). Increased ethylene evolution by plants exposed to various biotic and abiotic stresses has been widely documented (Moeder et al. 2002; Wang et al. 2002). Several authors (e.g. Moeder et al. 2002) have reported that the inhibition of ethylene biosynthesis results in a significant reduction of O3induced leaf lesion formation, ascribing to this hormone and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) a pivotal role in the complex signalling transduction pathways leading to cell death. More specifically, Diara et al. (2005) found differences in ethylene evolution in two poplar clones, “I-214” displaying earlier and less pronounced ethylene emission than “Eridano”. However, high ethylene evolution under Cd stress may be not perceived by plants, which would become relatively unresponsive to stress ethylene and show photosynthesis inhibition (Masood et al. 2012). Collectively, exposure to toxic metals is known to negatively affect plant traits, such as protein content and functionality, which drive fundamental metabolic pathways and defence mechanisms against oxidative stress (Lingua et al. 2012), thus potentially aggravating the consequences of air pollutants. In poplar, the effects of heavy metals and the ability of the plant to tolerate their toxicity have been widely studied (e.g. Di Baccio et al. 2009, 2011; Sebastiani et al. 2004; Tognetti et al. 2004; Borghi et al. 2007, 2008). Instead, the effect of metals on a plant’s ability to deal with other environmental stresses has received less attention (de Silva et al. 2012), while it is well known that the response of plants to a combination of stresses cannot be extrapolated from their responses to individual stress factors (Johnson et al. 2002; Mittler 2006). Studies on the possible interactions between Cd and O3 pollution in plants have been limited to herbaceous plants (Di Cagno et al. 2001; Li et al. 2011). In poplar, O3 and Cd stresses seem to share common toxicity pathways at physiological and molecular levels (Diara et al. 2005; Di Baccio et al. 2008; Kieffer et al. 2009; Pietrini et al. 2010), by affecting synergistically or antagonistically whole plant
2066
responses when they act concomitantly. On the other hand, a reduction in stomatal conductance and, consequently, water (and metal) uptake might provide a physiological basis for cross protection afforded against excess Cd by O3 treatment, and vice versa. Bohler et al. ( 2013) identified a set of proteins that changed similarly during O3 and drought stress, indicating cross talk in the molecular response of plants exposed to these stresses, while stomatal closure was of minor importance in the combined treatment. Castagna et al. (2013) studied the concomitant effects of O3 and Cd on poplar and found that Cd induced a reduction in stomatal conductance and a significant accumulation of H2O2 and nitric oxide (NO) in Eridano and I-214 clones and that Cd negatively affected the carotenoid contents in I-214. Here we predict that Cd and O3 share some common toxicity pathways and induce antagonistic rather than synergic responses. To test this hypothesis, we assayed stress indicators at the leaf level, including photosynthetic capacity, ethylene emission and oxidative status in these two poplar clones, differing for their sensitivity to O3. In particular, we studied whether assimilation and protection functions in leaves were differently influenced by the Cd or O3 alone or in combination. In addition, the possibility that O3 exposure may somehow interact with metal accumulation in leaves was evaluated by measuring Cd concentrations in leaves of control and O3-exposed plants.
Material and methods Plant material, Cd treatment and O3 fumigation Woody cuttings (30 cm in length) of two poplar clones, Populus x canadensis I-214 (O3-tolerant) and Populus deltoides×Populus maximowiczii Eridano (O3-sensitive), known for their differential responses to O3 in terms of development of leaf injuries following acute O3 exposure (Diara et al. 2005; Ranieri et al. 1999, 2000), were planted in 4-l plastic pots filled with soil (93.5 %:1.75 %:4.75 % sand/silt/ clay mix; organic carbon 2.34 %; inorganic carbon 0.096 %). Cuttings were irrigated, every 2–3 days (for a total of ten applications of approximately 50 ml solutions), with halfstrength Hoagland solutions added with 0 mM Cd(NO3)2 (−Cd) or 5 mM Cd(NO3)2 (+Cd) to reach the final total Cd concentration of 150 mg Cd kg−1 soil dw in the treated pot substrate (balanced with Ca(NO3)2 in the case of controls). After about 2 months (from cutting plantation) of outdoor cultivation (from May to July) under a shade net (mean daylight 500–600 μmol photons m−2 s−1) (43° 43′ N, 10° 23′ E, Pisa, Italy), plants were randomly selected and, for each clone and each Cd treatment, assigned to a control (charcoalfiltered air) or an O3-fumigation group. Plants were adapted to the growth chamber conditions for 48 h at a day/night temperature of 20/17 °C, relative humidity (RH) of 60–85 % and a
Environ Sci Pollut Res (2015) 22:2064–2075
14-h photoperiod at a photosynthetic photon flux density (PPFD) of 530 μmol m−2 s−1 from incandescent lamps. Six homogeneous (in size and structure) plants per clone were selected and then fumigated for 15 days with 60 nl l−1 O3 (5 h a day, from 08.00 to 13.00 h; referred to as O3-treated samples), or supplied with charcoal-filtered air under the same conditions (referred to as C samples). Ozone was generated by passing pure oxygen through a Fisher 500 air-cooled generator (Fisher Labor und Verfahrenstechnik, Meckenheim, Germany), and the O3 concentration of the fumigation chamber was continuously monitored with a UV analyser (Model 8810, Monitor Labs, San Diego, CA). During O3 fumigation, the temperature in the growth chambers (C and O3-treatment) was maintained at 20 ±1 °C and RH at 85±5 %, and PPFD at plant height was 530 μmol m−2 s−1. All measurements were made on three individual plants of each clone-treatment combination (n = 3) using fully expanded young leaves (LPI 4–6, Leaf Plastochron Index; Dickmann 1971). Determination of Cd concentration After harvesting, leaves were washed in distilled water. This plant material was then oven-dried at 70 °C until constant weight, ground to powder with liquid nitrogen and stored under vacuum until use. Samples (100 mg) were digested in 5 ml concentrated H2SO4, and hydrogen peroxide drops were added until their complete clarification. Final volumes were adjusted to 25 ml by ultra-pure water. The concentration of Cd was determined by atomic absorption using a Perkin–Elmer (Perkin-Elmer Analyst 100, Waltham, MA, USA) spectrophotometer, using a 0–3 ppm standard curve of commercial standard (Carlo Erba Reagents, Milan, Italy). Gas exchange analysis All measurements were carried out on control and treated plants at the end of the 15-day O3 exposure, with a photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA). The A/Ci curves were determined by measuring the response of net photosynthesis (A) to varying intercellular CO2 partial pressure (Ci). External CO2 partial pressures (Ca) were supplied in steps; the CO2 assimilation rate was first measured by setting the reference CO2 concentration near ambient (38 Pa) and then at 30, 20, 10, 5, 40 (two times), 60 and 80 Pa, with irradiance maintained at a saturating value of 500 μmol m−2 s−1 (considering growth chamber conditions). Measurements were recorded automatically at each Ca set point when photosynthesis had equilibrated. Measurements began after stomatal conductance (Gw) had reached maximum values. Foliage temperature during determination of the A/C i curves was maintained at 27 °C by means of
Environ Sci Pollut Res (2015) 22:2064–2075
thermoelectric coolers. Leaf-to-air vapour pressure deficit averaged 1.6 kPa. We analysed the A/Ci curves to estimate three biochemical parameters potentially limiting to photosynthesis: maximum rate of carboxylation (Vcmax, μmol m−2 s−1), light-saturated rate of electron transport (Jmax, μmol m−2 s−1) and rate of triose phosphate utilisation for sucrose and starch synthesis (TPU, μmol m−2 s−1). The CO2 compensation point (Гcomp, Pa) was calculated as the point that the model function intercepted the x-axis (CXX = 0). The daytime (mitochondrial) respiration (Rday, μmol m−2 s−1) or CO2 released in the light by processes other than photorespiration was also estimated. These variables were computed by the A/ Ci curve-fitting model developed by Farquhar et al. (1980), as in Marchi et al. (2008). We used the Michaelis-Menten constants of Rubisco used by Wullschleger (1993), where Kc (Michaelis-Menten constant for RuBP carboxylation) = 16 Pa, Ko (Michaelis-Menten constant for oxygenation)= 37,961 kPa and τ (specificity factor for Rubisco)=2823. The relative stomatal limitation to photosynthesis (Lg, %), an estimate of the proportion of the reduction in photosynthesis attributable to CO2 diffusion between the atmosphere and the site of carboxylation, was calculated from A/Ci curves by the method of Farquhar and Sharkey (1982) as Lg =(1−A/ Ao)*100, where A is the rate of net photosynthesis at the growth Ca (38 Pa) and Ao is the photosynthetic rate when Ci (38 Pa) equals the growth Ca. Under these conditions, Ao is the rate of photosynthesis that would occur if there were no diffusive limitations to CO2 transfer from the bulk atmosphere to the site of carboxylation. For this calculation, mesophyll conductance was considered infinitely large. The internal CO2 partial pressure (Ci, μmol mol−1), the net photosynthesis (Amax, μmol m−2 s−1) and the corresponding Gw were determined from A/Ci curves measured at saturating irradiance (for chamber conditions) and ambient CO2 partial pressure (Ca, about 380 μmol mol−1). Chlorophyll fluorescence analysis Chlorophyll fluorescence emissions in 60 min dark-adapted leaves (the same used for gas exchange analysis) were measured with a portable pulse amplitude modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany) to estimate the effect of treatments on photosystem II (PSII) efficiency. Background fluorescence signal (Fo), the maximum fluorescence (Fm) and potential quantum yield of PSII photochemistry [Fv/Fm =(Fm −Fo)/Fm] were determined. Ethylene determination Fifteen minutes after excision, leaves were incubated within sealed containers at room temperature; after 1 h, 2 ml samples were withdrawn with a hypodermic syringe. Ethylene
2067
evolution was measured by injecting samples into a gas chromatograph (HP5890, Hewlett-Packard, Menlo Park, CA, USA) equipped with a dual flame ionisation detector and a metal column (150×0.4 cm internal diameter) packed with alumina (70–230 mesh). The column and detector temperatures were 70 and 350 °C, respectively. Nitrogen (N2) was used as a carrier at a flow rate of 40 ml min−1 (Mensuali Sodi et al. 1992). Ascorbate determination Freshly harvested intact leaves (10 g) were rinsed with distilled water and vacuum-infiltrated (−65 kPa, three cycles of 30 s each) in 50 ml of 66 mM K-phosphate buffer (pH 7.0) and 100 mM KCl. After infiltration, the leaves were wiped and centrifuged (1,500×g for 10 min at 4 °C). The total amount of ascorbate (ASA) in intercellular washing fluid (IWF) was quantified immediately after its extraction to minimise the ascorbate oxidase (AAO)-dependent oxidation of ASA during measurements. The absence of such oxidation of ASA was confirmed by adding 10 mM sodium azide (an inhibitor of AAO) to the buffer used to obtain the extracellular leaf fluid (IWF). The quantitative determination was carried out according to Okamura (1980) and Law et al. (1983). An aliquot of IWF was added to 10 % TCA (w/v), and after addition of 5 M NaOH, the mixture was centrifuged at 12,000×g for 2 min. To quantify ASA, 150 mM phosphate buffer (pH 7.4) was added to the supernatant. Then, each sample was supplied with 10 % TCA (w/v), 44 % H3PO4 solution (v/v), 4 % α-α’-dipyridyl (w/v) in 70 % methanol and 3 % FeCl3 (w/v). The total amount of reduced (ASA) + oxidised (dehydroascorbate, DHA) forms was determined by incubating supernatant with 10 mM DTT, followed by the addition of 0.5 % of N-ethylmaleimide solution. Samples were then treated as for ASA determination. After vigorous stirring, all the samples (for ASA and ASA + DHA determinations) were kept at 37 °C for 60 min, and then the absorbance was read at 525 nm with a spectrophotometer (Ultrospec 2100 pro, GE Healthcare Europe GmbH, Milan, Italy) against a standard curve of pure ASA (Sigma) in the 0– 40 nmol range. DHA levels were determined by subtracting ASA from the total ascorbate content (ASA + DHA); the redox state of ascorbate was calculated as ASA/(ASA+ DHA)×100. The potentially available leaf antioxidant concentration per unit of O3 flux was calculated for each plant, following Di Baccio et al. (2008): AA=A/FO3, where AA represents the available amount of the antioxidant per unit of O3 flux, A is antioxidant concentration expressed on a leaf area basis, and FO3 (nmol m−2 s−1) is the stomatal O3 flux. We calculated FO3 as FO3 =Oa(GO3), where GO3 is stomatal conductance of water vapour multiplied by 0.613 and Oa is ambient O3 concentration (nl l−1). Because internal leaf O3 concentration approaches zero and the mesophyll resistance to O3 is typically
2068
small, we ignored internal leaf O3 concentration when calculating O3 flux. Statistical analysis The experiment was setup in a completely randomised design (n=3). The effects of clone (Eridano and I-214), Cd concentration in the substrate, O3 fumigation (charcoal-filtered air and O3 exposure) and of their interactions were evaluated by three-way ANOVA. The data were tested for homogeneity of variances by Barlett’s test, also sensitive to non-normal data prior to the application of statistical analysis. When the interaction among the three factors (clone × Cd × O3) was significant (P≤0.05), a post hoc test evaluating the separation of means was performed by Tukey’s HSD test at the 0.05 significance level. Data shown in tables and figures are means ± SE. Statistical analysis was conducted using STATISTICA 8.0 (StatSoft, Inc., Tulsa, OK).
Results Cadmium concentration in leaves The total amount of Cd in the native substrates (before Cd(NO3)2 addition) was below (0.17±0.04 ppm) the regulatory limit (2 ppm) specified by the Italian Environmental Legislation (d.lgs. 156/2006 and 4/2008). The two poplar clones did not differ in their ability to translocate Cd to leaves, the clone factor being not significant, according to the three-way ANOVA (Fig. 1). As expected, poplar growth in Cd-enriched soils led to a significant increase in Cd concentration in leaves. Cadmium concentration in leaves was also not significantly affected by O3 fumigation, although a tendency for lower metal concentration in O3-fumigated leaves of I-214 was observed.
Fig. 1 Concentration of Cd in leaves (mg kg−1 dw) of the hybrid poplar clones Eridano and I-214 treated with 0 (−Cd) or 150 (+Cd) mg Cd kg−1 soil and exposed for 15 days (5 h a day) to charcoal-filtered air (C) or 60 nl l−1 O3 (O3). Data represent the mean of three replicates ± SE. The three-way ANOVA P values for the effect of clone, Cd addition to soil, exposures to O3, and of their interactions are shown. A single asterisk indicates significance at P≤0.05, two asterisks at P≤0.01, and three asterisks at P≤0.001
Environ Sci Pollut Res (2015) 22:2064–2075
Photosynthesis parameters and chlorophyll fluorescence The photosynthetic capacity (Vcmax, Jmax and TPU) was not significantly affected by clone or Cd, while O3 had a significant effect on Vcmax and Jmax and less on TPU (P=0.066) (Table 1). The effect of O3 was generally towards reducing Vcmax and Jmax, while TPU tended to increase or remain stable; interactions among factors were never significant. The relationship between Jmax and Vcmax was linear through treatments and with similar slope across clones (y=4.9946x− 15.578, R2 =0.44) (Fig. 2). The overall ratio between Jmax and Vcmax averaged 4.62, without differences between clones or treatments. Again, R d a y ranged between 0.9 and 2.0 μmol m−2 s−1, without differences between clones or treatments (Table 1); interactions among factors were never significant. CO2 compensation point (Гcomp) was influenced by clone and O3 factor and their interaction (Table 1); in particular, Гcomp was generally higher in I-214 than in Eridano and increased in I-214 after O3 treatment. The effect of Cd and clone on Amax and Lg was not significant, while O3 had a significant and negative effect on Amax and positive on Lg (particularly in I-214) (Table 1). Interactions among factors were never significant for Amax, which was reduced by the only O3 factor. Conversely, Gw and Ci (and Ci/Ca) were affected by clone only, with generally higher values in Eridano (Table 1); the interaction clone × O3 was significant in the case of Gw and Lg. In general, Fv/Fm was affected by Cd, and only the interactions clone × O3 and clone × Cd were significant (Table 1). The time course measurements (see Supplementary material for additional descriptions) of gas exchange (after 1, 4, 8, 14 and 15 days of O3 exposure) did not show a definite pattern in photosynthetic activity at saturating light (Asat) and corresponding Gw. Eridano showed a significant decrease in Asat soon after 15-day O3 exposure period, which was not always present during the whole monitoring period; in I-214, Asat decreased after 8-day O3 exposure and recovered after 14 and 15 days (Supplementary Fig. S1). The behaviour of stomatal aperture resembled that of Asat, with a tendency to decrease Gw in Eridano and increase Gw in I-214, following O3 exposure. Minor variability between treatments and genotypes was also observed for Ci and Fv/Fm. However, a clear general reduction of Ci after 15-day O3 exposure, and Fv/Fm after 14- and 15-day O3 exposure, respectively, was observed (Supplementary Fig. S2). Ethylene emission In both clones, Cd and O3 had an opposite effect on ethylene emission—Cd decreasing and O3 increasing ethylene emission (Fig. 3). Ethylene emission differed between clones, with
Environ Sci Pollut Res (2015) 22:2064–2075
2069
Table 1 Net photosynthesis at saturating irradiance, considering the light intensity of the growth chamber (Amax, μmol CO2 m−2 s−1), stomatal conductance at corresponding irradiance (Gw, mol H2O m−2 s−1), internal CO 2 concentration (C i , ppm), mitochondrial respiration (R day, μmol m−2 s−1), maximum rate of carboxylation (Vcmax, μmol m−2 s−1), maximum rate of electron transport (Jmax, μmol m−2 s−1), rate of triose
Amax Gw Ci Rday Vcmax Jmax TPU Гcomp Lg Fv/Fm
phosphate utilisation (TPU, μmol m−2 s−1), CO2 compensation point (Гcomp, Pa), relative stomatal limitation to photosynthesis (Lg, %), and PSII potential quantum yield (Fv/Fm ratio) of the hybrid poplar clones Eridano and I-214 treated with 0 (−Cd) or 150 (+Cd) mg Cd kg−1 soil and exposed for 15 days (5 h a day) to charcoal-filtered air (control) or 60 nl l−1 O3
Eridano Control
Ozone
I-214 Control
Ozone
−Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd
13.38±0.38 11.06±0.46 0.338±0.007 0.324±0.112 300.8±3.5 299.2±14.6 0.91±0.16 1.49±0.36 43.68±2.61 46.08±9.44 267.8±14.1 188.1±38.3 6.56±0.75 5.21±0.32 5.91±0.13 6.65±0.40 28.81±2.16 27.38±6.86
10.36±1.08 9.70±0.51 0.252±0.049 0.248±0.055 288.8±12.9 295.3±15.7 1.20±0.46 1.38±0.38 37.60±1.61 40.19±7.78 157.1±26.9 134.2±9.9 6.04±0.47 8.73±2.19 6.61±0.72 6.80±0.64 30.88±2.78 30.76±4.09
11.76±0.54 12.72±0.54 0.099±0.013 0.129±0.022 267.1±7.4 277.1±7.0 1.60±0.33 1.39±0.66 51.23±6.46 65.57±2.38 247.3±48.6 294.7±85.4 4.88±0.17 4.38±0.38 6.48±0.34 5.88±0.52 23.33±4.37 15.94±2.40
9.26±0.94 9.43±1.09 0.192±0.029 0.176±0.021 288.4±3.3 281.0±0.8 1.96±0.51 1.60±0.44 39.57±2.17 41.67±4.72 184.3±20.3 227.7±78.8 7.61±2.49 5.75±0.91 8.46±1.03 9.55±1.15 40.00±1.79 36.55±3.96
−Cd +Cd
0.804±0.005 0.745±0.028
0.814±0.008 0.785±0.003
0.802±0.012 0.806±0.006
0.791±0.003 0.775±0.006
Clone 0.391 0.001*** 0.021* 0.211 0.063 0.151 0.290 0.039* 0.857 0.458
Cd 0.543 0.980 0.787 0.879 0.180 0.930 0.778 0.485 0.275 0.009**
Clone × Cd 0.109 0.818 0.930 0.298 0.465 0.177 0.319 0.828 0.409 0.035*
Clone × O3 0.683 0.049* 0.154 0.748 0.141 0.805 0.762 0.026* 0.010* 0.015*
Cd × O3 0.880 0.803 0.738 0.655 0.442 0.706 0.469 0.567 0.639 0.783
P value Amax Gw Ci Rday Vcmax Jmax TPU Гcomp Lg Fv/Fm
O3
RESEARCH ARTICLE
Effects of combined ozone and cadmium stresses on leaf traits in two poplar clones Antonella Castagna & Daniela Di Baccio & Anna Maria Ranieri & Luca Sebastiani & Roberto Tognetti
Received: 24 March 2014 / Accepted: 18 August 2014 / Published online: 30 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Information on plant responses to combined stresses such as ozone (O3) and cadmium (Cd) is scarce in tree species. On the other hand, high O3 concentrations in the atmosphere and heavy metal contaminations in water and soil simultaneously affect forest ecosystems. Toxic metals may exacerbate the consequences of air pollutants. In this research, two poplar clones, differently sensitive to O3 (“I-214” O3tolerant and “Eridano” O3-sensitive), were grown for 5 weeks in pots supplied with 0 and 150 mg Cd kg−1 soil and then exposed to a 15-day O3 fumigation (60 nl l−1, 5 h a day) or supplied with charcoal-filtered air under the same conditions (referred to as control samples). The effects of the two Responsible editor: Elena Maestri Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-3481-8) contains supplementary material, which is available to authorized users. A. Castagna (*) : A. M. Ranieri Dipartimento di Scienze Agrarie, Alimentari e Agro-Ambientali, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy e-mail: [email protected] D. Di Baccio : L. Sebastiani BioLabs, Istituto di Scienze della Vita, Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy R. Tognetti Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Contrada Fonte Lappone, 86090 Pesche, Italy R. Tognetti (*) The EFI Project Centre on Mountain Forests (MOUNTFOR), Via Edmund Mach 1, 38010 San Michele all’Adige, Italy e-mail: [email protected] Present Address: D. Di Baccio Istituto di Biologia Agroambientale e Forestale, Consiglio Nazionale delle Ricerche, Via Salaria km 29,300, 00015 Monterotondo Scalo, Italy
stressors, alone or in combination, on Cd accumulation, photosynthetic capacity, ethylene emission and oxidative state were investigated in fully expanded leaves. Cadmium accumulation in leaves caused a reduction, but not complete failure, of photosynthesis in Eridano and I-214 poplar clones. The reduction in assimilation rate was more important following O3 fumigation. Stomatal aperture after O3 treatment, instead, increased in I-214 and decreased in Eridano. Overall, Cd treatment was effective in decreasing ethylene emission, whereas O3 fumigation increased it in both clones, although interacting with the metal treatment. Again, O3 fumigation induced a significant increase in ascorbate (ASA) + dehydroascorbate (DHA) content, which was strongly oxidised by O3, thus decreasing the redox state. On the other hand, Cd treatment had a positive effect on ASA content and redox state in I-214, but not in Eridano. Although Cd and O3 are known to share some common toxicity pathways, the combined effects induced distinct clone-specific responses, underlying the complexity of plant reactions to multiple stresses. Keywords Ascorbic acid . Cd . Eridano . Ethylene . I-214 . O3 . Photosynthesis Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid Amax Net photosynthesis at saturating irradiance APX Ascorbate peroxidise ASA Ascorbate Cd Cadmium Ci Internal CO2 concentration DHA Dehydroascorbate Gw Stomatal conductance H2O2 Hydrogen peroxide NADPH Nicotinamide adenine dinucleotide phosphate NO Nitric oxide O3 Ozone
Environ Sci Pollut Res (2015) 22:2064–2075
[O3] PPFD ROS
O3 concentration Photosynthetic photon flux density Reactive oxygen species
Introduction Heavy metal pollution of soil and water has become a major worrying environmental issue worldwide, because of the contamination of large areas due to the anthropogenic activity (Pilon-Smits 2005). Among heavy metals, cadmium (Cd) is of particular concern due to its easy uptake by roots and high toxicity for plants (as well as animals). Cadmium can accumulate in roots up to micromolar concentration, before altering ultra-structural traits and browning root tips (Ederli et al. 2004). Being relatively mobile, part of the absorbed Cd can be translocated from roots and accumulated in shoots (Zacchini et al. 2011), causing chlorosis, inhibition of growth and even plant death, owing to its influence on stomatal behaviour, photosynthesis, respiration and water and nutrient uptake (e.g. Liu et al. 2011; Cocozza et al. 2014). Cadmium injury has been attributed to several factors, such as blocking of essential functional groups in biomolecules (Schützendübel et al. 2002) and displacement of metal ions from biomolecules (Rivetta et al. 1997). Cadmium may be involved in the suppression of antioxidant defence and in the overproduction of reactive oxygen species (ROS) (He et al. 2011; Iannone et al. 2010), which interfere with the redox status, possibly leading to uncontrolled oxidative stress (Rodríguez-Serrano et al. 2006). ROS increase could be indirectly caused by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in membranes (Romero-Puertas et al. 2004) and/or by reduction of activity/content of enzymatic and non-enzymatic antioxidants (Wójcik and Tukiendorf 2011). An open question is whether Cd accumulated in the foliage can have interactive effects with other factors of stress, eliciting ROS production. Tropospheric ozone (O3) is considered to be the most important air pollutant affecting vegetation (Ashmore 2005; Wittig et al. 2009). Prolonged exposures to relatively low O3 concentration [O3] (within the range of 20–50 nl l−1) affect plant health through stomatal uptake (Matyssek and Sandermann 2003; Panek 2004), causing leaf chlorosis (Ribas et al. 2005; Vollenweider et al. 2003), accelerated leaf senescence (Gielen et al. 2007; Zheng et al. 2002), reduced photosynthesis (Bagard et al. 2008; Di Baccio et al. 2008; Wittig et al. 2007) and decreased carbon allocation to sink tissues (Andersen 2003; Wittig et al. 2009), and affecting plant biomass and radial growth (Grulke et al. 2002; Panek and Goldstein 2001). In particular, leaf metabolism may be reoriented to repair processes and defence mechanisms (e.g. Cabané et al. 2004; Betz et al. 2009). Once O3 enters the leaf through stomata, it degrades rapidly in the apoplast to form various ROS (Langebartels et al. 2002; Vahala et al. 2003). Among ROS, the hydrogen
2065
peroxide (H2O2), in particular, is known to trigger a complex sequence of events leading to endogenous ROS production and activation of mitogen-activated protein kinase cascade, which in turn affects the synthesis of molecules such as ethylene, salicylic and jasmonic acids, involved in the signalling route and in modulation of the plant responses to the imposed stress (Kangasjärvi et al. 2005). However, when ROS production exceeds the plant ability to maintain the ROS concentration below a tolerance threshold, oxidative damage occurs (Castagna and Ranieri 2009). Among antioxidant metabolites, ascorbic acid, in addition to playing a key role in detoxification of H2O2 as a specific substrate for ascorbate peroxidase (APX), is capable of direct scavenging of O3 molecules and O3-derived ROS (Ranieri et al. 1999; Conklin and Barth 2004). Ethylene acts as a second messenger in oxidative signal transduction and regulates the induction and the spread of oxidative stress symptoms (Rao et al. 2002; Vahala et al. 2003). Increased ethylene evolution by plants exposed to various biotic and abiotic stresses has been widely documented (Moeder et al. 2002; Wang et al. 2002). Several authors (e.g. Moeder et al. 2002) have reported that the inhibition of ethylene biosynthesis results in a significant reduction of O3induced leaf lesion formation, ascribing to this hormone and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) a pivotal role in the complex signalling transduction pathways leading to cell death. More specifically, Diara et al. (2005) found differences in ethylene evolution in two poplar clones, “I-214” displaying earlier and less pronounced ethylene emission than “Eridano”. However, high ethylene evolution under Cd stress may be not perceived by plants, which would become relatively unresponsive to stress ethylene and show photosynthesis inhibition (Masood et al. 2012). Collectively, exposure to toxic metals is known to negatively affect plant traits, such as protein content and functionality, which drive fundamental metabolic pathways and defence mechanisms against oxidative stress (Lingua et al. 2012), thus potentially aggravating the consequences of air pollutants. In poplar, the effects of heavy metals and the ability of the plant to tolerate their toxicity have been widely studied (e.g. Di Baccio et al. 2009, 2011; Sebastiani et al. 2004; Tognetti et al. 2004; Borghi et al. 2007, 2008). Instead, the effect of metals on a plant’s ability to deal with other environmental stresses has received less attention (de Silva et al. 2012), while it is well known that the response of plants to a combination of stresses cannot be extrapolated from their responses to individual stress factors (Johnson et al. 2002; Mittler 2006). Studies on the possible interactions between Cd and O3 pollution in plants have been limited to herbaceous plants (Di Cagno et al. 2001; Li et al. 2011). In poplar, O3 and Cd stresses seem to share common toxicity pathways at physiological and molecular levels (Diara et al. 2005; Di Baccio et al. 2008; Kieffer et al. 2009; Pietrini et al. 2010), by affecting synergistically or antagonistically whole plant
2066
responses when they act concomitantly. On the other hand, a reduction in stomatal conductance and, consequently, water (and metal) uptake might provide a physiological basis for cross protection afforded against excess Cd by O3 treatment, and vice versa. Bohler et al. ( 2013) identified a set of proteins that changed similarly during O3 and drought stress, indicating cross talk in the molecular response of plants exposed to these stresses, while stomatal closure was of minor importance in the combined treatment. Castagna et al. (2013) studied the concomitant effects of O3 and Cd on poplar and found that Cd induced a reduction in stomatal conductance and a significant accumulation of H2O2 and nitric oxide (NO) in Eridano and I-214 clones and that Cd negatively affected the carotenoid contents in I-214. Here we predict that Cd and O3 share some common toxicity pathways and induce antagonistic rather than synergic responses. To test this hypothesis, we assayed stress indicators at the leaf level, including photosynthetic capacity, ethylene emission and oxidative status in these two poplar clones, differing for their sensitivity to O3. In particular, we studied whether assimilation and protection functions in leaves were differently influenced by the Cd or O3 alone or in combination. In addition, the possibility that O3 exposure may somehow interact with metal accumulation in leaves was evaluated by measuring Cd concentrations in leaves of control and O3-exposed plants.
Material and methods Plant material, Cd treatment and O3 fumigation Woody cuttings (30 cm in length) of two poplar clones, Populus x canadensis I-214 (O3-tolerant) and Populus deltoides×Populus maximowiczii Eridano (O3-sensitive), known for their differential responses to O3 in terms of development of leaf injuries following acute O3 exposure (Diara et al. 2005; Ranieri et al. 1999, 2000), were planted in 4-l plastic pots filled with soil (93.5 %:1.75 %:4.75 % sand/silt/ clay mix; organic carbon 2.34 %; inorganic carbon 0.096 %). Cuttings were irrigated, every 2–3 days (for a total of ten applications of approximately 50 ml solutions), with halfstrength Hoagland solutions added with 0 mM Cd(NO3)2 (−Cd) or 5 mM Cd(NO3)2 (+Cd) to reach the final total Cd concentration of 150 mg Cd kg−1 soil dw in the treated pot substrate (balanced with Ca(NO3)2 in the case of controls). After about 2 months (from cutting plantation) of outdoor cultivation (from May to July) under a shade net (mean daylight 500–600 μmol photons m−2 s−1) (43° 43′ N, 10° 23′ E, Pisa, Italy), plants were randomly selected and, for each clone and each Cd treatment, assigned to a control (charcoalfiltered air) or an O3-fumigation group. Plants were adapted to the growth chamber conditions for 48 h at a day/night temperature of 20/17 °C, relative humidity (RH) of 60–85 % and a
Environ Sci Pollut Res (2015) 22:2064–2075
14-h photoperiod at a photosynthetic photon flux density (PPFD) of 530 μmol m−2 s−1 from incandescent lamps. Six homogeneous (in size and structure) plants per clone were selected and then fumigated for 15 days with 60 nl l−1 O3 (5 h a day, from 08.00 to 13.00 h; referred to as O3-treated samples), or supplied with charcoal-filtered air under the same conditions (referred to as C samples). Ozone was generated by passing pure oxygen through a Fisher 500 air-cooled generator (Fisher Labor und Verfahrenstechnik, Meckenheim, Germany), and the O3 concentration of the fumigation chamber was continuously monitored with a UV analyser (Model 8810, Monitor Labs, San Diego, CA). During O3 fumigation, the temperature in the growth chambers (C and O3-treatment) was maintained at 20 ±1 °C and RH at 85±5 %, and PPFD at plant height was 530 μmol m−2 s−1. All measurements were made on three individual plants of each clone-treatment combination (n = 3) using fully expanded young leaves (LPI 4–6, Leaf Plastochron Index; Dickmann 1971). Determination of Cd concentration After harvesting, leaves were washed in distilled water. This plant material was then oven-dried at 70 °C until constant weight, ground to powder with liquid nitrogen and stored under vacuum until use. Samples (100 mg) were digested in 5 ml concentrated H2SO4, and hydrogen peroxide drops were added until their complete clarification. Final volumes were adjusted to 25 ml by ultra-pure water. The concentration of Cd was determined by atomic absorption using a Perkin–Elmer (Perkin-Elmer Analyst 100, Waltham, MA, USA) spectrophotometer, using a 0–3 ppm standard curve of commercial standard (Carlo Erba Reagents, Milan, Italy). Gas exchange analysis All measurements were carried out on control and treated plants at the end of the 15-day O3 exposure, with a photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA). The A/Ci curves were determined by measuring the response of net photosynthesis (A) to varying intercellular CO2 partial pressure (Ci). External CO2 partial pressures (Ca) were supplied in steps; the CO2 assimilation rate was first measured by setting the reference CO2 concentration near ambient (38 Pa) and then at 30, 20, 10, 5, 40 (two times), 60 and 80 Pa, with irradiance maintained at a saturating value of 500 μmol m−2 s−1 (considering growth chamber conditions). Measurements were recorded automatically at each Ca set point when photosynthesis had equilibrated. Measurements began after stomatal conductance (Gw) had reached maximum values. Foliage temperature during determination of the A/C i curves was maintained at 27 °C by means of
Environ Sci Pollut Res (2015) 22:2064–2075
thermoelectric coolers. Leaf-to-air vapour pressure deficit averaged 1.6 kPa. We analysed the A/Ci curves to estimate three biochemical parameters potentially limiting to photosynthesis: maximum rate of carboxylation (Vcmax, μmol m−2 s−1), light-saturated rate of electron transport (Jmax, μmol m−2 s−1) and rate of triose phosphate utilisation for sucrose and starch synthesis (TPU, μmol m−2 s−1). The CO2 compensation point (Гcomp, Pa) was calculated as the point that the model function intercepted the x-axis (CXX = 0). The daytime (mitochondrial) respiration (Rday, μmol m−2 s−1) or CO2 released in the light by processes other than photorespiration was also estimated. These variables were computed by the A/ Ci curve-fitting model developed by Farquhar et al. (1980), as in Marchi et al. (2008). We used the Michaelis-Menten constants of Rubisco used by Wullschleger (1993), where Kc (Michaelis-Menten constant for RuBP carboxylation) = 16 Pa, Ko (Michaelis-Menten constant for oxygenation)= 37,961 kPa and τ (specificity factor for Rubisco)=2823. The relative stomatal limitation to photosynthesis (Lg, %), an estimate of the proportion of the reduction in photosynthesis attributable to CO2 diffusion between the atmosphere and the site of carboxylation, was calculated from A/Ci curves by the method of Farquhar and Sharkey (1982) as Lg =(1−A/ Ao)*100, where A is the rate of net photosynthesis at the growth Ca (38 Pa) and Ao is the photosynthetic rate when Ci (38 Pa) equals the growth Ca. Under these conditions, Ao is the rate of photosynthesis that would occur if there were no diffusive limitations to CO2 transfer from the bulk atmosphere to the site of carboxylation. For this calculation, mesophyll conductance was considered infinitely large. The internal CO2 partial pressure (Ci, μmol mol−1), the net photosynthesis (Amax, μmol m−2 s−1) and the corresponding Gw were determined from A/Ci curves measured at saturating irradiance (for chamber conditions) and ambient CO2 partial pressure (Ca, about 380 μmol mol−1). Chlorophyll fluorescence analysis Chlorophyll fluorescence emissions in 60 min dark-adapted leaves (the same used for gas exchange analysis) were measured with a portable pulse amplitude modulation fluorometer (PAM-2000, Walz, Effeltrich, Germany) to estimate the effect of treatments on photosystem II (PSII) efficiency. Background fluorescence signal (Fo), the maximum fluorescence (Fm) and potential quantum yield of PSII photochemistry [Fv/Fm =(Fm −Fo)/Fm] were determined. Ethylene determination Fifteen minutes after excision, leaves were incubated within sealed containers at room temperature; after 1 h, 2 ml samples were withdrawn with a hypodermic syringe. Ethylene
2067
evolution was measured by injecting samples into a gas chromatograph (HP5890, Hewlett-Packard, Menlo Park, CA, USA) equipped with a dual flame ionisation detector and a metal column (150×0.4 cm internal diameter) packed with alumina (70–230 mesh). The column and detector temperatures were 70 and 350 °C, respectively. Nitrogen (N2) was used as a carrier at a flow rate of 40 ml min−1 (Mensuali Sodi et al. 1992). Ascorbate determination Freshly harvested intact leaves (10 g) were rinsed with distilled water and vacuum-infiltrated (−65 kPa, three cycles of 30 s each) in 50 ml of 66 mM K-phosphate buffer (pH 7.0) and 100 mM KCl. After infiltration, the leaves were wiped and centrifuged (1,500×g for 10 min at 4 °C). The total amount of ascorbate (ASA) in intercellular washing fluid (IWF) was quantified immediately after its extraction to minimise the ascorbate oxidase (AAO)-dependent oxidation of ASA during measurements. The absence of such oxidation of ASA was confirmed by adding 10 mM sodium azide (an inhibitor of AAO) to the buffer used to obtain the extracellular leaf fluid (IWF). The quantitative determination was carried out according to Okamura (1980) and Law et al. (1983). An aliquot of IWF was added to 10 % TCA (w/v), and after addition of 5 M NaOH, the mixture was centrifuged at 12,000×g for 2 min. To quantify ASA, 150 mM phosphate buffer (pH 7.4) was added to the supernatant. Then, each sample was supplied with 10 % TCA (w/v), 44 % H3PO4 solution (v/v), 4 % α-α’-dipyridyl (w/v) in 70 % methanol and 3 % FeCl3 (w/v). The total amount of reduced (ASA) + oxidised (dehydroascorbate, DHA) forms was determined by incubating supernatant with 10 mM DTT, followed by the addition of 0.5 % of N-ethylmaleimide solution. Samples were then treated as for ASA determination. After vigorous stirring, all the samples (for ASA and ASA + DHA determinations) were kept at 37 °C for 60 min, and then the absorbance was read at 525 nm with a spectrophotometer (Ultrospec 2100 pro, GE Healthcare Europe GmbH, Milan, Italy) against a standard curve of pure ASA (Sigma) in the 0– 40 nmol range. DHA levels were determined by subtracting ASA from the total ascorbate content (ASA + DHA); the redox state of ascorbate was calculated as ASA/(ASA+ DHA)×100. The potentially available leaf antioxidant concentration per unit of O3 flux was calculated for each plant, following Di Baccio et al. (2008): AA=A/FO3, where AA represents the available amount of the antioxidant per unit of O3 flux, A is antioxidant concentration expressed on a leaf area basis, and FO3 (nmol m−2 s−1) is the stomatal O3 flux. We calculated FO3 as FO3 =Oa(GO3), where GO3 is stomatal conductance of water vapour multiplied by 0.613 and Oa is ambient O3 concentration (nl l−1). Because internal leaf O3 concentration approaches zero and the mesophyll resistance to O3 is typically
2068
small, we ignored internal leaf O3 concentration when calculating O3 flux. Statistical analysis The experiment was setup in a completely randomised design (n=3). The effects of clone (Eridano and I-214), Cd concentration in the substrate, O3 fumigation (charcoal-filtered air and O3 exposure) and of their interactions were evaluated by three-way ANOVA. The data were tested for homogeneity of variances by Barlett’s test, also sensitive to non-normal data prior to the application of statistical analysis. When the interaction among the three factors (clone × Cd × O3) was significant (P≤0.05), a post hoc test evaluating the separation of means was performed by Tukey’s HSD test at the 0.05 significance level. Data shown in tables and figures are means ± SE. Statistical analysis was conducted using STATISTICA 8.0 (StatSoft, Inc., Tulsa, OK).
Results Cadmium concentration in leaves The total amount of Cd in the native substrates (before Cd(NO3)2 addition) was below (0.17±0.04 ppm) the regulatory limit (2 ppm) specified by the Italian Environmental Legislation (d.lgs. 156/2006 and 4/2008). The two poplar clones did not differ in their ability to translocate Cd to leaves, the clone factor being not significant, according to the three-way ANOVA (Fig. 1). As expected, poplar growth in Cd-enriched soils led to a significant increase in Cd concentration in leaves. Cadmium concentration in leaves was also not significantly affected by O3 fumigation, although a tendency for lower metal concentration in O3-fumigated leaves of I-214 was observed.
Fig. 1 Concentration of Cd in leaves (mg kg−1 dw) of the hybrid poplar clones Eridano and I-214 treated with 0 (−Cd) or 150 (+Cd) mg Cd kg−1 soil and exposed for 15 days (5 h a day) to charcoal-filtered air (C) or 60 nl l−1 O3 (O3). Data represent the mean of three replicates ± SE. The three-way ANOVA P values for the effect of clone, Cd addition to soil, exposures to O3, and of their interactions are shown. A single asterisk indicates significance at P≤0.05, two asterisks at P≤0.01, and three asterisks at P≤0.001
Environ Sci Pollut Res (2015) 22:2064–2075
Photosynthesis parameters and chlorophyll fluorescence The photosynthetic capacity (Vcmax, Jmax and TPU) was not significantly affected by clone or Cd, while O3 had a significant effect on Vcmax and Jmax and less on TPU (P=0.066) (Table 1). The effect of O3 was generally towards reducing Vcmax and Jmax, while TPU tended to increase or remain stable; interactions among factors were never significant. The relationship between Jmax and Vcmax was linear through treatments and with similar slope across clones (y=4.9946x− 15.578, R2 =0.44) (Fig. 2). The overall ratio between Jmax and Vcmax averaged 4.62, without differences between clones or treatments. Again, R d a y ranged between 0.9 and 2.0 μmol m−2 s−1, without differences between clones or treatments (Table 1); interactions among factors were never significant. CO2 compensation point (Гcomp) was influenced by clone and O3 factor and their interaction (Table 1); in particular, Гcomp was generally higher in I-214 than in Eridano and increased in I-214 after O3 treatment. The effect of Cd and clone on Amax and Lg was not significant, while O3 had a significant and negative effect on Amax and positive on Lg (particularly in I-214) (Table 1). Interactions among factors were never significant for Amax, which was reduced by the only O3 factor. Conversely, Gw and Ci (and Ci/Ca) were affected by clone only, with generally higher values in Eridano (Table 1); the interaction clone × O3 was significant in the case of Gw and Lg. In general, Fv/Fm was affected by Cd, and only the interactions clone × O3 and clone × Cd were significant (Table 1). The time course measurements (see Supplementary material for additional descriptions) of gas exchange (after 1, 4, 8, 14 and 15 days of O3 exposure) did not show a definite pattern in photosynthetic activity at saturating light (Asat) and corresponding Gw. Eridano showed a significant decrease in Asat soon after 15-day O3 exposure period, which was not always present during the whole monitoring period; in I-214, Asat decreased after 8-day O3 exposure and recovered after 14 and 15 days (Supplementary Fig. S1). The behaviour of stomatal aperture resembled that of Asat, with a tendency to decrease Gw in Eridano and increase Gw in I-214, following O3 exposure. Minor variability between treatments and genotypes was also observed for Ci and Fv/Fm. However, a clear general reduction of Ci after 15-day O3 exposure, and Fv/Fm after 14- and 15-day O3 exposure, respectively, was observed (Supplementary Fig. S2). Ethylene emission In both clones, Cd and O3 had an opposite effect on ethylene emission—Cd decreasing and O3 increasing ethylene emission (Fig. 3). Ethylene emission differed between clones, with
Environ Sci Pollut Res (2015) 22:2064–2075
2069
Table 1 Net photosynthesis at saturating irradiance, considering the light intensity of the growth chamber (Amax, μmol CO2 m−2 s−1), stomatal conductance at corresponding irradiance (Gw, mol H2O m−2 s−1), internal CO 2 concentration (C i , ppm), mitochondrial respiration (R day, μmol m−2 s−1), maximum rate of carboxylation (Vcmax, μmol m−2 s−1), maximum rate of electron transport (Jmax, μmol m−2 s−1), rate of triose
Amax Gw Ci Rday Vcmax Jmax TPU Гcomp Lg Fv/Fm
phosphate utilisation (TPU, μmol m−2 s−1), CO2 compensation point (Гcomp, Pa), relative stomatal limitation to photosynthesis (Lg, %), and PSII potential quantum yield (Fv/Fm ratio) of the hybrid poplar clones Eridano and I-214 treated with 0 (−Cd) or 150 (+Cd) mg Cd kg−1 soil and exposed for 15 days (5 h a day) to charcoal-filtered air (control) or 60 nl l−1 O3
Eridano Control
Ozone
I-214 Control
Ozone
−Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd −Cd +Cd
13.38±0.38 11.06±0.46 0.338±0.007 0.324±0.112 300.8±3.5 299.2±14.6 0.91±0.16 1.49±0.36 43.68±2.61 46.08±9.44 267.8±14.1 188.1±38.3 6.56±0.75 5.21±0.32 5.91±0.13 6.65±0.40 28.81±2.16 27.38±6.86
10.36±1.08 9.70±0.51 0.252±0.049 0.248±0.055 288.8±12.9 295.3±15.7 1.20±0.46 1.38±0.38 37.60±1.61 40.19±7.78 157.1±26.9 134.2±9.9 6.04±0.47 8.73±2.19 6.61±0.72 6.80±0.64 30.88±2.78 30.76±4.09
11.76±0.54 12.72±0.54 0.099±0.013 0.129±0.022 267.1±7.4 277.1±7.0 1.60±0.33 1.39±0.66 51.23±6.46 65.57±2.38 247.3±48.6 294.7±85.4 4.88±0.17 4.38±0.38 6.48±0.34 5.88±0.52 23.33±4.37 15.94±2.40
9.26±0.94 9.43±1.09 0.192±0.029 0.176±0.021 288.4±3.3 281.0±0.8 1.96±0.51 1.60±0.44 39.57±2.17 41.67±4.72 184.3±20.3 227.7±78.8 7.61±2.49 5.75±0.91 8.46±1.03 9.55±1.15 40.00±1.79 36.55±3.96
−Cd +Cd
0.804±0.005 0.745±0.028
0.814±0.008 0.785±0.003
0.802±0.012 0.806±0.006
0.791±0.003 0.775±0.006
Clone 0.391 0.001*** 0.021* 0.211 0.063 0.151 0.290 0.039* 0.857 0.458
Cd 0.543 0.980 0.787 0.879 0.180 0.930 0.778 0.485 0.275 0.009**
Clone × Cd 0.109 0.818 0.930 0.298 0.465 0.177 0.319 0.828 0.409 0.035*
Clone × O3 0.683 0.049* 0.154 0.748 0.141 0.805 0.762 0.026* 0.010* 0.015*
Cd × O3 0.880 0.803 0.738 0.655 0.442 0.706 0.469 0.567 0.639 0.783
P value Amax Gw Ci Rday Vcmax Jmax TPU Гcomp Lg Fv/Fm
O3
