Responses of Populus×euramericana (P. deltoides×P. nigra) clone Adda to increasing copper concentrations

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Environmental and Experimental Botany 61 (2007) 66–73

Responses of Populus × euramericana (P. deltoides × P. nigra) clone Adda to increasing copper concentrations M. Borghi a,∗ , R. Tognetti b , G. Monteforti a , L. Sebastiani a a

b

Scuola Superiore Sant’Anna, BioLabs, Piazza Martiri della Libert`a 33, I-56127, Pisa, Italy Universit`a degli Studi del Molise, Dipartimento di Scienze e Tecnologie per l’Ambiente e il Territorio (STAT), Contrada Fonte Lappone, I-86090 Pesche (IS), Italy Received 1 June 2006; received in revised form 9 February 2007; accepted 31 March 2007

Abstract We investigated the effect of high copper (Cu) concentrations on poplar woody cuttings (Populus × euramericana) clone Adda in order to estimate the degree of metal tolerance. Metal accumulation was also investigated to determine where and to what extent Cu is stored within the plant. Plant responses to Cu were determined comparing biomass growth and photosynthetic potential measured at concentrations of 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu supplied with Hoagland’s solution in an aerated hydroponic system. Results obtained in this study show that increasing levels of Cu resulted in a general reduction of plant growth at concentrations equal or higher than 100 ␮M. At these concentrations of Cu, reductions of chlorophyll contents and photosynthetic parameters were also observed. The metal was mainly accumulated in the root system at all Cu levels. Since no significant differences were noticed in growth and photosynthesis between control plants and those treated with 20 ␮M of Cu, we conclude that Adda clone was able to tolerate quite high concentration of Cu and we propose it as a good candidate for screening Cu tolerance in the field. © 2007 Elsevier B.V. All rights reserved. Keywords: Growth analysis; Photosynthesis; Poplar

1. Introduction Copper (Cu) is an essential micronutrient for plant, acting as a cofactor of enzymes, structural proteins and phytohormone receptors. However, when present in elevated concentrations it inhibits plant growth and development (Weng et al., 2005; Xu et al., 2006). Contamination of soils and ground water with Cu mainly results from human activities such as smelting, disposal of sewage sludge and agricultural practices (Nriagu and Pacyna, 1988; Epstein and Bassein, 2001; Brun et al., 2003). In addition, low price fertilizers mainly deriving from intensive farming (e.g. pig slurry) are widely used for non-food plantations and notoriously contain high amount of Cu (Giardini, 2002). Plants have evolved different strategies to cope with heavy metal stress, including detoxification and exclusion (Hall, 2002). Hyperaccumulators that naturally absorb high levels of metal ions in their shoots, possess multiple mechanisms of resistance,

∗

Corresponding author. Tel.: +39 050 883000; fax: +39 050 883495. E-mail address: [email protected] (M. Borghi).

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.03.001

and have a significant phytoremediation potential (Salt et al., 1998). Excluder species that prevent metal uptake into the roots, even though having low potential for metal extraction, can be successfully used to stabilize the soil and avoid further contamination by leaching and erosion (Peer et al., 2005). Responses of plants to high levels of Cu have been mainly investigated in herbaceous and ruderal species (Reichmann, 2002; Brun et al., 2001, 2003; Sgherri et al., 2001; Demirevska-Kepova et al., 2004; Cuypers et al., 2005), although the use of trees in managing contaminated sites has been attracting an increasing interest (Pulford and Watson, 2003). Multipurpose tree plantations bring environmental benefits in the process of reclamation of polluted soils (Coleman et al., 1995), and among them fast-growing hybrid poplars have been indicated as good candidates for stabilizing metal-contaminated sites (Djingova et al., 1999; Robinson et al., 2000; Sebastiani et al., 2004; Peuke and Rennenberg, 2005). In this sense, most research works deal with accumulation capacity and biomass production of poplars exposed to high concentrations of pollutants while the knowledge of physiological mechanisms of heavy metal accumulation and tolerance is

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still in its infancy (Arisi et al., 2000; Rennenberg and Will, 2000; Koprivova et al., 2002; Di Baccio et al., 2003, 2005; Tognetti et al., 2004). The success of using poplars for environmental restoration will depend on several factors, including the extent of soil contamination, metal availability and plant ability to intercept, absorb and accumulate metals in shoots (Sebastiani et al., 2004). Factors such as the extent of metal tolerance are highly variable among Populus species and clones, due to abundant genetic variation throughout the genus (Brunner et al., 2004). Comparative studies on metal accumulation and growth demonstrated that different clones respond differently to the exposure to the same industrial waste or contaminants (Sebastiani et al., 2004; Tognetti et al., 2004). The necessity for large-scale experiments in open conditions testing the degree of metal resistance in poplar plantations, reinforce the need for preliminary screenings in controlled environments to examine physiological processes of tolerance in specific genotypes. In the present study we investigated the effect of increasing Cu concentrations on growth traits and photosynthetic parameters in Populus × euramericana (Dode) Guinier (Populus deltoides × P. nigra), clone Adda commercialized in Northern Italy (Fossati et al., 2005) in order to evaluate the tolerance to the metal. Uptake of metal was also examined to determine where and to what extent Cu is stored within the plant. Our results indicate that the Adda clone was resistant to quite high Cu concentrations; hence we propose this clone for experimental plantations to evaluate its potential use in environmental restoration of metal-contaminated sites.

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constant weight. Samples of root were extensively rinsed with deionised water soon after harvesting. Stem length (SL) and basal diameter (SD) were measured weekly on each plant. Leaf area (LA) was determined by the regression equation y = 0.7121x (R2 = 0.99) forced through the origin, where (y) is leaf area and (x) the product of the two leaf main orthogonal diameters. The equation was previously determined by measuring the surface area of 215 randomly selected leaves and analysed using the software Scion Image 4.0.2. (Scion Corporation, MD, USA). Specific leaf area (SLA) was calculated as the ratio of leaf area to corresponding leaf dry mass. Relative growth rate (RGR) was calculated as RGR = [loge MT − loge MT0 ]/Tx − T0 ] where MT is the total dry mass at sampling time Tx (T1 and T2 ) and MT0 is the initial (T0 ) total plant dry mass average (Hunt, 1978). 2.3. Copper and nitrogen determinations Samples of leaves, stem and roots were separately ovendried at 70 ◦ C and ground to a powder in a laboratory mill. 0.5 g of plant material was mineralised in HCl–HNO3 (dilution factor 3:1), clarified with ultra pure water and used for measurements of total Cu in an atomic absorption spectrophotometer (Atomic Absorption Spectrometer 373, Perkin-Elmer, Norwalk, CT, USA). Organic nitrogen (N) content was determined in leaves by using the Kijeldhal method. 2.4. Gas exchange and chlorophyll fluorescence measurements

2. Materials and methods 2.1. Plant material and treatments Poplar woody cuttings (P. × euramericana, Adda clone) were rooted and grown in pots containing a universal loam:perlite mixture (1:1, v:v), and daily irrigated with deionised water for about 30 days. Plants were successively transferred in a growth chamber (temperature 23 ◦ C; relative humidity 55–75% night–day; photoperiod 16-h light:8-h dark) and grown in pots filled with clay (diameter 13–15 mm, pH 7.0) suitable for hydroponic culture, and supplied with Hoagland’s solution, pH 6.7 (Arnon and Hoagland, 1940). To prevent the death of roots the solution was completely replaced every 7 days and aerated by diffused air aeration systems. After 15 days of acclimation to the hydroponic system, 40 homogeneous plants were selected and randomly assigned to five groups of treatment with Hoagland’s solution containing different concentrations of CuSO4 ·5H2 O: 0.4 (control), 20, 100, 500 and 1000 ␮M. Four plants randomly selected at the beginning of the experiment were used to determine the initial biomass (T0 ). 2.2. Growth analysis Fifteen (T1 ) and 34 (T2 ) days from the beginning of the experiment four plants from each of five groups of treatment were randomly selected and separated into leaves, petioles, stem, roots and woody cutting, weighted and oven-dried at 70 ◦ C until

The CO2 response curves (A/Ci ) were measured on the fourth and fifth leaf from the apex, using an infrared gas analyzer LI-6400 (LI-Cor, Lincoln, NE, USA). Leaf chamber temperature and humidity were adjusted to maintain a leaf-to-air vapour pressure difference of about 1.3 kPa. The CO2 response curves were obtained by changing the CO2 concentration entering the cuvette from 50 to 800 ␮mol mol−1 by means of an external CO2 cartridge mounted on the LI-6400 console and automatically controlled by a CO2 injector. Light intensity was maintained at 600 ␮mol photons m−2 s−1 . The response of leaf A to Ci was analysed according to the mechanistic model of CO2 assimilation proposed by Farquhar (Farquhar et al., 1980). The biochemical model describing A calculates three parameters potentially limiting photosynthesis: Vcmax (maximum carboxylation rate of ribulose-1,5-bisphosphate carboxylase–oxygenase, Rubisco), Jmax (ribulose-1,5-bisphosphate, RuBP, regeneration capacity mediated by electron transport rate) and TPU (rate of triose phosphate utilization for sucrose and starch synthesis). Day respiration rate is designated Rday and results from processes other than photorespiration. The CO2 compensation point (Γ c ) was calculated as the point where the model function crosses the x-axis (A = 0). The model was parameterised and fitted to the experimental data using Photosyn Assistant (Dundee Scientific, Dundee, Scotland, UK). Contemporary to gas exchange determinations, chlorophyll fluorescence emissions in 30-min dark-adapted leaves were measured with a portable pulse amplitude modulation fluorome-

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ter (PAM-2000, Walz, Effeltrich, Germany) to estimate the effect of treatments on efficiency of PSII. The background fluorescence signal (Fo ), the maximum fluorescence (Fm ), and the potential quantum yield of PSII photochemistry [Fv /Fm = (Fm − Fo )/Fm ] were determined.

of Cu treatment at different concentrations. Statistical analysis was conducted using the software CoStat 6.2 (CoHort Software, CA, USA). Separation of means was performed using LSD test at P = 0.05 significance level. 3. Results

2.5. Chlorophyll determination 3.1. Growth parameters Chlorophyll a and b contents were determined by reading the absorbance at 664 and 647 nm of the extracts obtained from two disks of 10 mm in diameter from the fourth to fifth leaf from the apex. The extraction was performed with N,Ndimethylformamide (1:20, w:v) for 48 h at 4 ◦ C in the dark and chlorophyll content determined according to Moran (1982). Spectrophotometer measurements were monitored on a Lambda 25 Spectrophotometer (Perkin-Elmer).

Biomass of all plant organs (leaves, petioles, stem, and roots) increased at time T2 in comparison with corresponding values measured at time T1 (Table 1). At time T2 differences in biomass of all organs between plants grown at 0.4 (control) and 20 ␮M of Cu were not significant while at 100, 500 and 1000 ␮M of Cu a general reduction of plant biomass was noticed. Trend of growth reduction at increasing Cu was reconfirmed by RGR values. Values of SL, SD and LA were also affected by treatment with Cu at concentrations higher than 20 ␮M (Table 2). SLA was similar among treatments indicating that the reduction of LA at the highest Cu concentrations matches with the decrease of leaf biomass caused by progressive shedding.

2.6. Statistical analysis The experiment was set up in a completely randomised design with four replicates for time T1 and T2 . Data were subjected to a one-way analysis of variance (ANOVA) to examine the effect

Table 1 Leaves, petioles, stem, roots and total biomass of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 15 (T1 ) or 34 (T2 ) days Time

Plant organ

Cu treatment

ANOVA P-values

0.4 ␮M

T1

T2

Leaves Petioles Stem Roots Total Leaves Petioles Stem Roots Total

2.59 0.19 0.87 0.37 4.04 7.56 0.71 3.38 1.24 12.90

± ± ± ± ± ± ± ± ± ±

20 ␮M 0.64 0.07 0.36 0.09 1.17 3.10 a 0.29 a 1.67 a 0.44 a 5.50 a

3.14 0.28 1.22 0.40 5.05 8.10 0.75 3.65 1.08 13.59

± ± ± ± ± ± ± ± ± ±

100 ␮M 1.26 0.15 0.63 0.17 2.23 1.80 a 0.12 a 0.77 a 0.14 ab 2.84 a

2.28 0.15 0.63 0.24 3.32 3.83 0.35 1.55 0.72 6.46

± ± ± ± ± ± ± ± ± ±

500 ␮M 0.65 0.05 0.30 0.10 1.12 0.65 b 0.06 b 0.33 b 0.09 bc 1.15 bc

2.27 0.16 0.740 0.42 3.60 3.07 0.27 1.23 0.70 5.28

± ± ± ± ± ± ± ± ± ±

1000 ␮M 0.86 0.07 0.43 0.35 1.73 0.42 b 0.03 b 0.07 b 0.22 c 0.75 c

1.29 0.07 0.33 0.15 1.85 2.23 0.18 0.82 0.43 3.67

± ± ± ± ± ± ± ± ± ±

0.193 0.02 0.08 0.06 0.37 0.50 b 0.05 b 0.33 b 0.18 c 1.07 c

ns ns ns ns ns .0003 .0001 .0006 .0026 .0003

Values are the mean ± standard deviation (n = 4). Units are g of dry mass. ANOVA P-values for Cu treatments are shown. Means in rows followed by different letters (a–c) are significantly different at P = 0.05 level (LSD test). Table 2 Stem length (SL), stem diameter (SD), leaf area (LA), specific leaf area (SLA), relative growth rate (RGR) of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 15 (T1 ) or 34 (T2 ) days Time

Growth parameter

Cu treatment 0.4 ␮M

T1

T2

SL SD LA SLA RGR SL SD LA SLA RGR

0.36 5.19 0.08 0.03 0.04 0.64 6.98 0.23 0.03 0.06

± ± ± ± ± ± ± ± ± ±

ANOVA P-values 20 ␮M

0.07 ab 0.63 a 0.01 ab 0.00 ab 0.01 a 0.10 a 1.13 a 0.07 a 0.00 0.01 a

0.41 6.01 0.11 0.03 0.05 0.72 7.73 0.27 0.03 0.06

± ± ± ± ± ± ± ± ± ±

100 ␮M 0.07 a 0.99 a 0.03 a 0.00 a 0.02 a 0.02 a 0.40 a 0.03 a 0.00 0.00 a

0.29 5.09 0.06 0.02 0.03 0.45 5.79 0.11 0.02 0.04

± ± ± ± ± ± ± ± ± ±

500 ␮M 0.06 bc 0.25 a 0.02 bc 0.00 c 0.01 a 0.03 b 0.55 b 0.01 b 0.00 0.00 bc

0.29 4.93 0.06 0.02 0.04 0.37 5.51 0.08 0.02 0.03

± ± ± ± ± ± ± ± ± ±

1000 ␮M 0.06 bc 0.78 a 0.02 bc 0.00 bc 0.02 a 0.01 bc 0.30 b 0.00 b 0.00 0.00 cd

0.20 3.83 0.03 0.02 0.01 0.29 4.79 0.05 0.02 0.02

± ± ± ± ± ± ± ± ± ±

0.03 c 0.74 b 0.00 c 0.00 c 0.00 b 0.05 c 0.70 b 0.01 b 0.00 0.00 d

.0034 .0122 .0070 .0007 .0236 .0000 .0002 .0000 ns .0001

Values are the mean ± standard deviation (n = 4). Units are m (SL), mm (SD), m2 (LA), m2 g−1 (SLA) and days−1 (RGR). ANOVA P-values for Cu treatments are shown. Means in rows followed by different letters (a–d) are significantly different at P = 0.05 level (LSD test).

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Fig. 1. Copper concentration in leaves (a), stem (b) and roots (c) of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 34 (T2 ) days. Units are ppm. Values are mean ± standard deviation (n = 4). The P-values of ANOVA for Cu treatments are shown. Bars with different letters are significantly different at P = 0.05 level (LSD test).

At time T1 the only evident toxic effects were at 1000 ␮M of Cu. 3.2. Copper and nitrogen contents Copper concentration in leaves (Fig. 1a) was significantly higher in plants grown at 20 ␮M of Cu (10.2 ppm) than in control plants (7.0 ppm) while diminished to 7.4 ppm in plants grown at 1000 ␮M of Cu. Copper concentration in stem was similar in control as well as in treated plants (Fig. 1b). Cu concentration in plants grown at 20 ␮M (7.1 ppm) was significantly different only from plants treated with 1000 ␮M (5.2 ppm). In roots (Fig. 1c)

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Fig. 2. Total Cu content in leaves (a), stem (b) and roots (c) of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 34 (T2 ) days. Units are mg. Values are mean ± standard deviation (n = 4). The Pvalues of ANOVA for Cu treatments are shown. Bars with different letters are significantly different at P = 0.05 level (LSD test).

Cu concentration was about 27 times higher than in leaves and stem, and increased progressively at raising of Cu along the treatments. Total Cu content measured in leaves, stem and roots was tightly dependent on treatment (Fig. 2). In leaves of plants grown with 20 ␮M of Cu the total content of the metal was higher (0.08 mg) than in control plants (0.05 mg) however quickly decreased at raising of Cu in Hoagland’s solution. Differences of Cu content in stem were between control and plants treated with 100 ␮M of Cu or higher, while no differences were observed between control and plants treated with 20 ␮M of Cu. In roots total Cu content was 4 and 20 times higher than in leaves and

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Table 3 Nitrogen concentration (N) and total nitrogen content (N total) in leaves of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 34 (T2 ) days Nitrogen content

N (%) N total

Cu treatment

ANOVA P-values

0.4 ␮M

20 ␮M

100 ␮M

500 ␮M

1000 ␮M

3.47 ± 0.23 a 26.24 ± 11.09 a

3.11 ± 0.14 b 25.10 ± 4.99 a

3.07 ± 0.18 b 11.72 ± 1.82 b

2.82 ± 0.00 bc 8.71 ± 1.33 b

2.69 ± 0.16 c 5.97 ± 1.26 b

.0001 .0002

Values are the mean ± standard deviation (n = 4). Units are expressed as N w:w% or mg of total N, respectively. ANOVA P-values for Cu treatments are shown. Means in rows followed by different letters (a–c) are significantly different at P = 0.05 level (LSD test). Table 4 Chlorophyll a (Chla ), b (Chlb ), total (Chltot ) and chlorophyll a/b ratio (Chla/b ) in leaves of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 34 (T2 ) days Chlorophyll parameter

Cu treatment 0.4 ␮M

Chla Chlb Chltot Chla/b

0.101 0.027 0.128 3.69

ANOVA P-values 20 ␮M

± ± ± ±

0.007 c 0.002 a 0.009 b 0.13 b

0.111 0.029 0.141 3.78

± ± ± ±

100 ␮M 0.019 bc 0.005 a 0.024 ab 0.10 b

0.121 0.032 0.145 3.74

500 ␮M

± ± ± ±

0.006 ab 0.002 a 0.008 a 0.12 b

0.127 0.032 0.159 4.09

± ± ± ±

1000 ␮M 0.025 a 0.009 a 0.034 a 0.38 a

0.013 0.003 0.016 3.89

± ± ± ±

0.003 d 0.000 b 0.004 c 0.47 ab

.0000 .0000 .0000 .0099

Values are the mean ± standard deviation (n = 4). Units of chlorophyll are expressed as ␮g mm−2 . ANOVA P-values for Cu treatments are shown. Means in rows followed by different letters (a–d) are significantly different at P = 0.05 level (LSD test).

stems, respectively, and it was strongly increased along the treatments. Percentage of N in leaves decreased progressively from about a maximum of 3.5% in control plants to a minimum of 2.7% measured in plants treated with 1000 ␮M of Cu (Table 3). Total N content in leaves showed a strong reduction starting from treatment with 100 ␮M (55% lower than control) while differences between control and plants grown with 20 ␮M of Cu were not significant. 3.3. Chlorophyll concentration Chlorophyll a content showed a progressive increase at raising of Cu from 0.4 to 500 ␮M nevertheless a strong reduction was observed at 1000 ␮M (87% lower than control) (Table 4). Chlorophyll b and total chlorophyll content showed a similar trend although differences in chlorophyll b content between controls and plants treated with 500 ␮M of Cu were not statis-

tically significant. The chlorophyll a/b ratio increased in plants grown with 500 ␮M of Cu, while no difference was evident among controls and plants grown with 20, 100 or 1000 ␮M of Cu. 3.4. Gas exchange and chlorophyll fluorescence Values of Vcmax and Jmax were similar among treatments in the range from 0.4 to 500 of Cu (Table 5). A noticeable decrease in averaged values of photosynthetic potentials was evident at the concentration of 1000 ␮M of Cu. Other photosynthetic parameters, TPU and Γ c , followed a similar trend, though statistically not significant. Values of Rday and Jmax /Vcmax ratio were relatively stable throughout all treatments. Potential quantum yield of PSII photochemistry was generally rather low, averaging 0.6, regardless of the treatment. Indeed, increasing Cu levels did not induce a significant variation in Fv /Fm values (Table 5).

Table 5 Photosynthetic parameters from A/Ci curves and chlorophyll fluorescence in leaves of P. × euramericana clone Adda treated with 0.4 (control), 20, 100, 500 and 1000 ␮M of Cu for 34 (T2 ) days Photosynthetic parameter

Cu treatment 0.4 ␮M

Rday Vcmax Jmax TPU Γc Jmax /Vcmax Fv /Fm

0.68 39.83 89.63 3.64 5.42 2.35 0.63

± ± ± ± ± ± ±

ANOVA P-values 20 ␮M

0.38 15.73 ab 23.33 ab 1.09 0.61 0.50 0.06

0.88 47.39 104.64 4.27 5.49 2.24 0.62

100 ␮M ± ± ± ± ± ± ±

0.37 19.30 ab 42.19 a 1.20 1.83 0.72 0.05

1.07 48.11 107.67 5.64 5.57 2.36 0.61

± ± ± ± ± ± ±

500 ␮M 0.43 24.26 a 40.95 a 3.41 1.43 0.89 0.05

0.74 28.87 65.10 3.06 5.95 2.40 0.60

± ± ± ± ± ± ±

1000 ␮M 0.84 16.26 bc 36.35 bc 1.21 2.23 0.77 0.04

0.69 16.64 40.38 2.48 3.89 2.52 0.60

± ± ± ± ± ± ±

0.40 16.19 c 38.57 c 1.65 2.75 0.69 0.03

ns .0079 .0031 ns ns ns ns

Values are the mean ± standard deviation (n = 4). Units are expressed as ␮mol CO2 m−2 s−1 for Vcmax , TPU, and Rday , ␮mol electrons m−2 s−1 for Jmax , Pa for Γ c . ANOVA P-values for Cu treatments are shown. Means in rows followed by different letters (a–c) are significantly different at P = 0.05 level (LSD test).

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4. Discussion Sites contaminated by high levels of Cu are often located in the proximity of extraction mines (Mebane, 1997) or disposal sites (EPA, 1989). Contamination of agricultural soils by spraying of pesticides products, livestock manures and other organic wastes has also been reported (Delas, 1963; Labrecque et al., 1995; Nicholson et al., 1999). Cu is ubiquitous in sewage sludges, biosolids and wastewaters from industrial (e.g. tanneries), agricultural (liquid manure from intensive farming) and domestic sources that are utilized as low price fertilizers for bioenergy plantations (Rønde and Lepp, 1997). Growth inhibition, leaf area reduction and the decrease of root biomass, often accompanied by changes in root morphology, are the main symptoms of Cu toxicity (Lidon and Henriques, 1993; Maksymiec, 1997). Nevertheless, the degree of growth reduction and metal accumulation in response to Cu are strictly dependent on species and clones (Borgeg˚ard and H˚akan, 1989; Dickinson et al., 1992; Punshon et al., 1995), and physiological effects need to be considered before planning in situ plantings. We focused our research work on hybrid P. × euramericana, clone Adda commercialized and cultivated in Northern Italy (Fossati et al., 2005). In this region, poplar is mainly cultivated in wide riparian banks along Po River and in the streamside of other narrow rivers and canals. Plants grown on riparian strips play essential roles in restoration of aquatic and riparian systems (Naiman and D’Ecamps, 1997), but often deal with organic and inorganic pollutants. In the present experiment P. × euramericana clone Adda showed a reduction in all growth parameters after 34 days of treatment at Cu concentrations of 100 ␮M or more. Because root biomass reduction was observed as well, we supposed that the general decline of plant growth possibly reflects difficulties in nutrients uptake due to toxicity-induced small size and/or less efficient root apparatus. Reduced build up of nutrients to leaves was indicated by the strong decrease in total N contents, starting from the treatment with 100 ␮M of Cu. Kidd et al. (2004) observed inhibition of root elongation in Cistus grown in hydroponics only at Cu concentrations of 50 and 75 ␮M, while at Cu concentrations of 15 (control) and 25 ␮M no differences were observed. Plants treated with 20 ␮M of Cu did not show any decrease in growth potential compared to the control, thus demonstrating the capacity of P. × euramericana clone Adda to tolerate fairly high levels of Cu. At Cu concentrations of 20 ␮M, both content and concentration of metal increased in leaves and we did not observe any significant difference in Cu concentrations between leaves of control plants and those treated with 100 ␮M of Cu or more. The relatively high chlorophyll content and photosynthetic potential of plants grown at 20 ␮M of Cu could support this hypothesis. Punshon and Dickinson (1997) reported for Salix species the magnitude of Cu accumulation in decreasing order: roots, wood, new stem and leaves. In agreement with other studies on woody plants (Punshon et al., 1995; Rosselli et al., 2003) we found that Cu uptake into aerial tissues of Adda clone was relatively limited and most of Cu was localized in the root system. In roots copper is mainly bound to carboxylic groups of pectins and polygalacturonic acid and to N containing groups of proteins and enzymes

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of cell wall (Marschner, 1995). Alternatively to storage mechanisms in root tissues, the low Cu transport to aboveground plant organs could be due to binding of metal to the xylem vessel surfaces (Nissen and Lepp, 1997). Poplar has a large root apparatus capable to deepen and explore profound layers of ground, able to bind high amount of Cu (Fig. 2c) and for this reason it could be a good candidate for phytostabilization of Cu contaminated sites. Relatively high Cu concentrations have also been reported for roots of Betula (Kozlov et al., 1995; Maurice and Lagerkvist, 2000), Salix (Punshon and Dickinson, 1997) and Fraxinus (Arduini et al., 1998) exposed to gradually increasing concentrations of metal, along with a low aboveground translocation. Greger and Landberg (1999) noticed that Cu concentrations in roots of Salix viminalis L. clones were at least 30 times greater than in shoots, indicating poor translocation potentials. Excess Cu in the growth substrate generally induces a decrease in total chlorophyll content, which has been associated with the destruction of inner structure of chloroplasts and modifications of the lipid–protein composition of thylakoid membranes (Maksymiec, 1997). These phenotypes are thought to depend on local iron (Fe) deficiency owing to high levels of Cu, since Fe is necessary for chlorophyll biosynthesis and photosynthetic activity (Abad´ıa et al., 1989; P¨atsikk¨a et al., 2002). Nevertheless, an increase in photosynthetic pigments (Romeu-Moreno and Mas, 1999) and no effects on net photosynthesis (Rousos et al., 1989) have been reported in some plant species exposed to high Cu levels. Clone Adda displayed symptoms of foliar chlorosis restricted to old leaves at Cu concentrations of 100 and 500 ␮M, and to all leaves (young and old) at the highest concentration (1000 ␮M). Besides, photosynthetic parameters showed a threshold-type response: symptoms of a decreased photosynthetic efficiency were observed only at Cu concentration of 1000 ␮M. Indeed, the photochemical efficiency of PSII was never impaired by Cu treatments. Tognetti et al. (2004) observed that woody cuttings of hybrid poplar clone Eridano (P. deltoides × maximowiczii) grown for a season in soil amended with nutrient-rich organic material from tanneries (containing potentially toxic amounts of Zn, Cu, Cr and Cd) differed from control plants in chloroplast and stomatal limitations to CO2 assimilation, while this was not the case of clone I-214 (P. × euramericana). Photosynthetic capacity of both clones increased significantly in response to soil amendment: TPU, Rday , and Γ c were not affected by soil treatment, while Vcmax and Jmax were linearly related across treatments. Similarly, in the present experiment we observed that Jmax and Vcmax were linearly related across treatments (Jmax = 17.63 + 1.77Vcmax ; R2 = 0.77, P < 0.0001, regression not shown). Therefore, clone Adda, even at high Cu levels, was able to optimise resources allocation to preserve a balance between enzymatic capacity (Rubisco) and light-harvesting potential (chlorophyll) (Wullschleger, 1993). Nevertheless, photosynthetic potential and gas exchange might have been affected by Cu-induced N limitation (Pettersson and McDonald, 1994). An increased amount of pigments on a leaf area basis accompanied by a reduced expansion of foliage area has been reported as an index of Cu toxicity (Maksymiec, 1997). We did not

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observe any reduction in leaf area or significant variation in growth parameters in plants grown at 20 ␮M of Cu in comparison with control. Consequently, we inferred that clone Adda could tolerate Cu concentrations as higher as 20 ␮M, corresponding to more than 50 times the level of control in this experiment. The privileged accumulation of heavy metals in harvesting organs (i.e. woody structures of stem and branches) rather than in leaves, which return metals to ground annually due to shedding, is an important selective criterion for a phytoremediator species. However, avoidance of contaminant leakage is also advisable in all situations in which a resolutive approach to soil remediation have to be taken. For this purpose plants with wide expanse root apparatus and traits of tolerance are preferred. In this experiment we noticed that clone Adda accumulated Cu mainly in the root apparatus and it showed a quite wide tolerance to high Cu concentrations. Growing plants in solution cultures to screen for metal resistance has been successfully used (Punshon et al., 1995), and results obtained in hydroponics have been often confirmed in field experiments (Kopponen et al., 2001). Clone Adda demonstrated a fairly high Cu tolerance with the present growing system, where all Cu ions were by far available to plant uptake, which warrants further testing in field trials. Acknowledgements We thank the Istituto Sperimentale per la Pioppicoltura (CRA-MIPAF, Casale Monferrato, Italy) for providing cuttings of P. × euramericana, clone Adda. We are grateful to Ivan R. Baxter for critically reading the manuscript. This research was supported by a grant from Italian MUR (PRIN) and by funds of the Sant’Anna University. References Abad´ıa, A., Lemoine, Y., Tr´emoli`eres, A., Ambard-Bretteville, F., R´emy, R., 1989. Iron deficiency in pea: effects on pigment, lipid and pigment-protein complex composition of thylacoyds. Plant Physiol. Biochem. 27, 679–687. Arduini, I., Godbold, D.L., Onnis, A., Stefani, A., 1998. Heavy metals influence mineral nutrition of tree seedlings. Chemosphere 36, 739–744. Arisi, A.C.M., Mocquot, B., Lagriffoul, A., Mench, M., Foyer, C.H., Jouanin, L., 2000. Responses to cadmium in leaves of transformed poplars overexpressing ␥-glutamylcysteine synthetase. Physiol. Plant. 109, 143–149. Arnon, D.I., Hoagland, D.R., 1940. Crop production in artificial culture solutions and in soils with special reference to factors influencing yields and absorption of inorganic nutrients. Soil Sci. 50, 463–483. Borgeg˚ard, S.O., H˚akan, R., 1989. Biomass, root penetration and heavy metal uptake in birch in a soil cover over copper tailings. J. Appl. Ecol. 26, 585–595. Brun, L.A., Le Corff, J., Maillet, J., 2003. Effects of elevated soil copper on phenology, growth and reproduction of five ruderal plant species. Environ. Pollut. 122, 361–368. Brun, L.A., Maillet, J., Richarte, J., Herrmann, P., Remy, J.C., 2001. Relationships between extractable copper, soil properties and copper uptake by wild plants in vineyard soils. Environ. Pollut. 102, 151–161. Brunner, A.M., Busov, V.B., Strauss, S.H., 2004. Poplar genome sequence: functional genomics in an ecologically dominant plant species. Trends Plant Sci. 9, 49–56. Coleman, M., Dickinson, R., Isebrands, J., Karnoski, D., 1995. Carbon allocation and partitioning in aspen clones varying in sensitivity to tropospheric ozone. Tree Physiol. 15, 593–604.

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