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Silva Balcanica, 15(1)/2014

PHOTOSYNTHESIS AND GROWTH RESPONSE OF TWO PAULOWNIA HYBRID LINES TO HEAVY METALS Cd, Pb AND Zn

Kameliya Miladinova1, *Yuliana Markovska2, Nikolina Tzvetkova3, Katya Ivanova2, Mariya Geneva4,Teodora Georgieva1

1BioTree Ltd - Sofia2Faculty of Biology, Sofia University

3Faculty of Forestry, University of Forestry - Sofia4Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences - Sofia

Abstract

Paulownia tomentosa x fortunei (TF 01) and Paulownia elongata x fortunei (EF 02) hybrid lines exposed to different concentrations of Cd, Pb and Zn in hydroponic experiment were analyzed with reference to study the metal’s effects on the growth and photosynthetic parameters. Maximal accumulation of heavy metals occurred in the roots. These treatments caused significant reduction in total dry biomass, leaf area, stomatal conductance and tran-spiration rate, but increased leaf area ratio (LAR), net photosynthetic rate and water use efficiency. The accumulation of heavy metals in both lines increased in order Pb < Zn < Cd, while bioaccumulation coefficients (BC) rose in order Pb < Cd < Zn. The BC changed in different manner with increasing concentration of heavy metals in nutrient solution. The bioaccumulation coefficients of Pb and Cd were greater than 100. These results confirm both lines as accumulators which grow rapidly and have a potential for phytoremediation of metal contaminated soils showing phytostabilization mechanism.

Key words: heavy metals, gas exchange, growth, PaulowniaAbbreviations: gs – stomatal conductance; PN – net photosynthetic rate; E – transpi-

ration rate; WUE – water use efficiency.

INTRODUCTION

Increasing emissions of heavy metals are dangerous because they may get into the food chain with risk for human health (Galloway et al., 1982; Angelone, Bini, 1992). Cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) are some of the most widespread heavy metal contaminants of soils and act toxic in animals and plants at elevated concentrations (Kabata-Pendias, Pendias, 1991). Three different molecu-lar mechanisms of metal toxicity are induced as: heavy metals bind to the oxygen, nitrogen and sulphur atoms in the plant tissues: 1) production of reactive oxygen species by autooxidation and Fenton reaction; 2) blocking of essential functional groups in biomolecules; 3) displacement of essential metal ions from biomolecules (Schűtzendubel, Polle, 2002). The physiological effects of heavy metals on plants in-

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cluded: 1) perturbation of stomatal functions to changes in water relations and rate of gas exchange (Papadakis et al., 2004); 2) reduction of photosynthetic pigments and activity (Alaoui-Sossé et al., 2004; Lou et al., 2004); 3) disruption of the integrity of cellular membranes (Hall, 2002).

Phytoextraction is an environmentally friendly in situ technique for remedia-tion of heavy metal contaminated soils which involves the removal of toxins by the roots of the plants with subsequent transport to aerial plant organs. Plant bio-mass production and metal concentration in the biomass are important factors for the practical efficiency of phytoextraction (McGrath, Zhao, 2003). One strategy is to use high biomass crop plants like Brassica juncea, Helianthus annuus, Zea mays, Nicotiana tabacum (Meers et al., 2005; Tassi et al., 2007) or hyper-accumulator plants (Baker et al., 2000; Robinson et al., 2000). Widely used for phytoremediation of contaminated soils herbaceous hyper-accumulators are slow-growing plants and low-biomass producers which accumulate only one specific element and possess low depth roots. Deeper pollution and contamination caused by a number of metals re-quire using as an alternative fast-growing woody species with deep root system and the ability to grow on nutrient-poor soil. Some of them (poplar, willow, black locust, ash, or alder) are successfully used for remediation of substrates contaminated by inorganic and organic pollutants. Poplar and willow are mainly used for remediation of Cd polluted soils (Robinson et al., 2000; Lei et al., 2007), but their heavy metal tolerance is limited (Dietz, Schnoor, 2001). Populus nigra has shown to be useful as a biomonitor in heavy metal contaminated regions (Djingova et al., 1995) and now Populus has been internationally accepted as a model system for physiological and molecular tree studies (Tuscan et al., 2006). Paulownia is a promising woody spe-cies for the remediation of Pb, Zn, Cu and Cd polluted soils owing to its very high biomass productivity, rather than its metal accumulation potential (Doumett et al., 2008; 2011; Wang et al., 2010).

Paulownia is native from the China. P. tomentosa has been introduced into USA and Europe as an ornamental plant and is still widely used for this purpose. Trees introduced in Bulgaria reach 12 m average height and 13.4 cm average diam-eter during 7 years (Kalmukov, 1995). The project ‘Establishment of geographical plantations of Paulownia elongata hybrids in Bulgaria’ - Contract 37 with State Agency of Forests (2007-2010) was developed (Gyuleva, 2008). The correlation between Zn accumulation in the leaves and photosynthetic rate during adaptation at low CO2 conditions is established (Lazova et al., 2003). Over the last two de-cades Paulownia species has been extensively studied due to its ability to uptake nitrates and land contaminants, namely heavy metals (Doumett et al., 2008; 2011; Wang et al., 2010). This high-yielding tree can be used for the production of en-ergy, paper pulp and wooden building materials. Research on in vitro propagation of P. elongata and P. fortunei has been reported by Bergmann et al. (1997), Gyu-leva, Garelkova (1993) and Gyuleva (2010). Application of this technology for micropropagation of tree species offers a rapid means of producing clonal planting

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stock for afforestation, woody biomass production and it is effective way to main-tain the genetic gain (Park, Bonga, 1992). The use of nodal segments for in vitro propagation promoted a higher rate of multiplication of P. tomentosa (Ozaslan et al., 2005).

Plants used in the current paper are propagated and rooted according technol-ogy registered of BioTree Ltd., Bulgaria. The company’s laboratory is the largest producer and supplier of genetically superior Paulownia tissue-cultures – in vitro seedlings. The farmers prefer P. tomentosa x fortunei line TF 01 due to fast develop-ment and uniform regular growth. P. elongata x fortunei line EF 02 is less branchy and is used for the purpose of wood material production. There is no information about its tolerance to heavy metal stress and possibilities of management of soil pol-lution with these lines.

In this research, the effects of Cd, Pb and Zn on growth and photosynthetic gas exchange of Paulownia tomentosa x fortunei line TF 01 and Paulownia elongata x fortunei line EF 02, grown in hydroponic after transplant the explants are compared so as to provide fundamental base for vegetation restoration in contaminated soils.

MATERIALS AND METHODS

Plant materialExplants from the species P. tomentosa, P. elongata and their hybrids with P.

fortunei were used for developing of in vitro multiplication protocol. The explants were washed with Tween and subsequently sterilized with 0.1% mercury chloride (HgCl2) solution for 5 min and then rinsed tree times in sterile distilled water for 5, 10 and 15 min. For induction of shoots, explants were cultured on Murashige et Skoog’s (MS) nutrient medium (1962) supplemented with 2.5% (w/v) sucrose and 0.8% (w/v) agar. For shoot multiplication the medium was supplemented with 4.439 µM 6-benzylaminopurine (ВАР) and 0.537 µM 3-indolylacetic acid (IAA). The medium included 3% sucrose (w/v), 0.8% agar and vitamins. After multipli-cation the shoots were transferred to rooting medium based on half strength basal salts MS medium, 2% sucrose, 6% agar and vitamins supplemented with 4.92 µM IBA and 1.075 µM IAA. The pH of all media was adjusted to 5.7 using 0.1 N HCl and 0.1 N NaOH before autoclaving. All cultures were incubated under controlled conditions – 16 h photoperiod, light intensity of 35 μmol m-2 s-1 and 24/18±1ºC day/night temperature. After three weeks of rooting the shoots were washed from the medium with tap water and the roots were rinsed with 1.5 mL cm-3 Proplant solution.

Design, growth conditions and Cd, Pb, Zn treatment The experiments were set as ten treatments including control, each treatment

with 3 replications. The uniform plants were selected and transplanted to polyethyl-ene vessels containing 1.2 cm3 of 1/4 diluted Hellriegel (1898) solution with an ad-

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dition of A-Z microelements after Hoagland (pH 5.9) in growth chamber with a 16-h photoperiod (PAR 100 μmol m-2 s-1 on the upper leaf surface, 25/23±1ºC day/night temperature, relative humidity 60/70%). Each vessel contained two plants which represented one replication. After 21 days of cultivation the plants were transferred to 1/2 diluted Hellriegel solution with the addition of A-Z microelements (pH 5.9). The heavy metal treatment was applied on the 48th day after transplanting when the plants had adapted to the conditions of 1/2 diluted Hellriegel nutrient solution and one of the following heavy metals: Cd (0.5; 2.5 and 5.0 mg L-1 of Cd supplied as Cd(NO3)2.4H2O: MERCK); Pb (5.0; 10.0 and 20.0 mg L-1 of Pb supplied as Pb(NO3)2: MERCK) and Zn (10.0; 20.0 and 30.0 mg L-1 of Zn supplied as Zn(NO3)2.6H2O: MERCK) was added. Plants grown in nutrient solution without metals served as con-trols. The solutions were aerated every day and were changed every 3 d to prevent depletion of nutrients and heavy metals. Plants were harvested after 10 d of treat-ment. At the end of the experiment the plant samples were collected, washed with tap water and rinsed with distilled water before being separated into leaf, petiole, stem and root and fresh mass of each plant sample were measured gravimetrically. Dry mass of shoots and roots were determined after oven-drying (60oC) for 2 d until constant weight was obtained. Leaf area was calculated using software SigmaScan Pro 5. Leaf area ratio (LAR) was described as the leaf area (cm2) divided by the total plant dry weight (Hunt, 1982).

Analysis of heavy metals Total metal content in each organ was analyzed after sample homogenization

in a Blender. Dry vegetal tissues (0.5 g) were treated with 2 mL of 30% hydrogen peroxide and 3 mL 65% nitric acid and 9 mL of 37% hydrochloric acid (Doumett et al., 2008). The samples were heated on a heating block at 200oC to evaporate to dry-ness. The residue was taken up in 25 mL of 1 N HCl. Metal concentration were deter-mined on the inductively – coupled Plasma Mass Spectrometer (CCD Simultaneus ICP OES, Varian, Australia). The metal content in shoots was calculated on the basis of the relative dry mass (DM) of each aerial part of plants ([HM]leaves x DMleaves) + ([HM]stems x DMstems)/(DMleaves + DMstems) (Tassi et al., 2007). Since stems mainly act as a transport organ of heavy metal from the roots to the upper parts, above results in terms of leaves accumulation will be stressed, as such results are more suitable for comparison with the various treatments. The BC was calculated as the heavy metal content in plant divided by heavy metal concentration in the solution (Nanda – Ku-mar et al., 1995).

Gas-exchange measurementNet photosynthetic rate (PN, μmol CO2 m

-2 s-1), transpiration rate (E, mmol H2O m-2 s-1), and stomatal conductance (gs, mmol H2O m-2 s-1) were measured simul-taneously on 10th day after heavy metal treatments using a Portable gas analyzer Li-6400, (Li-Cor Inc., Lincoln, NE, USA), with a red-blue LED light source. Measure-

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ments were made in the late morning hours between 10:00 a.m. and 12:00 a.m. under standardized conditions (air temperature of 25.0oC, air humidity of 50% inside the gas exchange cuvette, 1000 μmol m-2 s-1 PAR and 350 μmol ambient concentration of CO2. Three to five fully developed, attached leaves of plants at each heavy metal treatment were measured and averaged. The following index was calculated: water use efficiency (WUE, μmol mmol-1 = PN E

-1 (Nijs et al., 1997).

Statistical evaluation Data are expressed as means ±SE, where n=5. Comparison of means was per-

formed by Fisher’s LSD test (P ≤ 0.05) after performing ANOVA analysis (Statgraph-ics Plus, v. 2.1). For the analysis of gas-exchange parameters and the water use effi-ciency a two-way ANOVA was used, with metal and Paulownia line as independent variables (Statistics: SYSTAT, v.7.0 (SPSS Inc., Chicago, USA, 1996).

RESULTS

Biomass productionVisual assessment of plants treated with heavy metals did not show any phyto-

toxic symptoms and treated plants had the same appearance as control plants. Mean values of total dry weight determined in Paulownia hybrid lines 58 days after trans-planting of plants to the condition of diluted Hellriegel nutrient solution and 10 days after treatment with heavy metals are reported in Table 1 and 2. Total dry weight for each individual line confirmed the strong biomass reduction after treatment with heavy metals, excepting variant with 0.5 mg L-1 Cd for EF 02. Root/shoot dry bio-mass ratio increased after all treatments in comparison with control. Enhancement is slightly for EF 02.

Mean values of leaf area in response to the type and concentration of heavy metals followed the same trend as total dry weights. Leaf area ratios (LARs) showed the capability of a plant in forming of photosynthetic surface and increased slightly after treatment with the highest concentrations of Cd and Pb, but not of Zn.

Heavy metal accumulationThe concentrations of heavy metals in different parts of the plants are

shown in Table 3 and 4. The highest accumulation of Cd, Pb and Zn was found in roots. In general, the metal content in plants increased with the increase in metal concentrations in solution, and the metal accumulation in roots was always significantly higher than that in stems and leaves. Highest values for Cd were obtained in the roots of EF 02 at 2.5 mg L-1 Cd, while similar content of Cd is established in the roots of TF 01 at 5.0 mg L-1 Cd. P. tomentosa x fortunei line TF 01 accumulated approximately 2-fold most Pb and Zn in the roots than P. elongata x fortunei line EF 02. The accumulation of heavy metals in both lines increased in order Pb < Zn < Cd, while BC rise in order Pb < Cd < Zn. The BC

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changed in different manner with increasing concentration of heavy metals in nutrient solution. The highest ones for Pb at 20.0 mg L-1 Pb (398.9 and 211.6), and for Zn at 10.0 mg L-1 Zn (80.7 and 43.9) were established for both lines. The highest BC for Cd at 2.5 mg L-1 (222.3) for line TF 01 and at 5.0 mg L-1 (113.3) for line EF 02 is obtained too.

PN and gas-exchange parametersThe addition of heavy metals in the nutrient solution enhanced photo-

synthetic rate (PN) in both lines compared to control (Fig. 1A, B, C), whereas stomatal conductance (gS) and transpiration rate (E) decreased sharply with an arising of metal concentrations with a minimum at the first (Pb) and the second (Cd and Zn) stage of the stress (Fig. 2A, B, C, 3A, B, C). At all of heavy metal treatments a significant increase in water use efficiency (WUE) was established, compared to control (Fig. 4A, B, C). WUE reached maximal values yet at the lowest (Cd and Pb) and the middle (Zn) heavy metal concentrations. In general, the values of all parameters measured for TF 01 were enhanced than that of EF 02 irrespecive of highest BC. Interestingly, the values of PN measured for both lines were smallest at highest values of BC. Two-way ANOVA revealed sig-nificant differences between the two Paulownia hybrid lines (Fs – line effect) in relation to all heavy metal effects (Fm – Cd, Pb or Zn effect) and line x metal interaction effect (Fs x Fm).

Table 1. Total dry biomass, root/shoot biomass ratio, total leaf area and LAR of P. tomentosa x fortunei line TF 01 grown in hydroponic and exposed 10 days to different concentrations of Cd, Pb and

Zn. Data are presented as means of five replications ± standard error. Different letters indicate significant differences assessed by Fisher’s LSD test (P ≤ 0.1) after performing of one-way ANOVA analysis.

Treatments (mg L-1)

Total dry mass (g) Root/shoot DM Total leaf area

(cm2) LAR (cm2 g-1)

Control 0.908±0.050b 0.232 429±16b 497±25a

0.5 Cd 0.455±0.045a 0.326 218±24a 474±57ab2.5 Cd 0.419±0.032a 0.328 195±40a 473±42a5.0 Cd 0.427±0.060a 0.349 207±38a 510±35b5.0 Pb 0.483±0.066a 0.328 241±39a 523±27a10.0 Pb 0.521±0.070a 0.327 257±39a 531±29a20.0 Pb 0.497±0.053a 0.359 241±33a 504±33a10.0 Zn 0.445±0.063a 0.349 198±27a 472±22a20.0 Zn 0.597±0.075a 0.315 277±38a 487±32a30.0 Zn 0.553±0.055a 0.329 268±27a 498±35a

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DISCUSSION

The effects of metals (especially Cd, Pb and Zn) on the structure and function of trees are still intensively studied. The roots of four willow and two poplar species responded to Cd treatment more sensitively than the shoots. Cd ions accumulated in roots suppressed their growth in all tested species (Šottnikova et al., 2003). The main morphological and structural effects of Cd on roots was summarized by Barcelò, Poschenrieder (1999): decreasing of root elongation and biomass, root tip damage, collapsing of root hairs or decreasing of their number, increasing or decreasing of lateral root formation. Our results showed that root elongation and biomass of P. tomentosa x fortunei plants were slightly suppressed, namely with the increase in Cd, Pb and Zn concentrations in solution (Miladinova et al., 2012a,b), but strong reduction of total plant dry biomass after all treatments is established (Table 1, 2). Willow and poplar species growing directly in Cd(NO3)2 showed better adaptation of roots than these growing in Knop nutrient solution and transferred to Cd(NO3)2. The positive effect of indirect treatment on pigment and starch contents, net photo-synthetic rate and specific leaf mass were reported (Lunačkova et al., 2003). These results showed that Cd impact depended on the cultivation conditions. The inhibition of primary and secondary growth as well as of the net photosynthetic rate were es-tablished in Populus tremula x P. alba genotype 717-1B4 at exposure to 360 mg Cd kg-1 soil. Cd is accumulated predominantly in the woody parts of stem, whereas Zn is localized in the leaves and had no effect on the growth (Durand et al., 2010). It was established a slight decrease of stem elongation and dry biomass in both Paulownia

Table 2. Total dry biomass, root/shoot biomass ratio, total leaf area and leaf area ratio (LAR) of P. elongata x fortunei line EF 02 grown in hydroponic and exposed 10 days to different concentra-

tions of Cd, Pb and Zn. Data are presented as means of five replications ± standard error. Different letters indicate significant differences assessed by Fisher’s LSD test (P ≤ 0.1) after performing of

one-way ANOVA analysis.

Treatments (mg l -1)

Total dry mass (g) Root/shoot DM Total leaf area

(cm2) LAR (cm2 g-1)

Control 1.233±0.092bc 0.196 502±26d 415±54ab0.5 Cd 1.298±0.041c 0.214 462±26cd 484±27abc2.5 Cd 0.929±0.043abc 0.258 376±36bcd 437±53abc5.0 Cd 0.486±0.065ab 0.226 271±48abc 560±73c5.0 Pb 0.785±0.030abc 0.234 357±42abcd 480±88abc10.0 Pb 0.581±0.037abc 0.240 303±19abcd 433±35abc20.0 Pb 0.287±0.016a 0.199 143±17a 530±72bc10.0 Zn 1.228±0.189c 0.217 534±21bcd 415±32a20.0 Zn 0.511±0.020ab 0.242 225±29ab 451±62abc30.0 Zn 0.409±0.021abc 0.227 171±25abcd 419±29abc

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Fig. 1. Net-photosynthesis rate (PN) in the mature leaves of P. tomentosa x fortunei line TF 01 and P. elongata x fortunei line EF 02, grown in hydroponic and exposed 10 days to different concentra-

tions of Cd (A), Pb (B) and Zn (C).

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hybrid lines with increase in heavy metal concentration of solution. The number of leaves and their dry biomass were lowered after treatments with increasing concen-trations of Cd and Pb, but these parameters increased in addition of Zn (Miladinova et al., 2012a,b). Leaf areas decreased strongly after all treatments, excepting EF 02 at 10 mg L-1 Zn (Table 2). Leaf area ratios (LARs) were also calculated (Table 1, 2) for the evaluating the capability of plant to invest biomass in the photosynthetic surface. Interestingly, the treatment with the highest concentration of Cd and Pb showed one of the highest values in spite of the lower total dry weight of both lines. The need of an improved leaf surface per dry weight unit is probably related to an increased pho-tosynthetic efficiency. Root/shoot dry biomass ratio confirmed the above-mentioned results (Table 1, 2). Plants treated with increasing concentrations of heavy metals had the highest investment in root biomass compared to the aerial section.

The effects of heavy metals on plants are complicated and related to many factors including ion type, concentrations and combinations of elements, plant de-velopmental stage and nutrient conditions (Broadly et al., 2007).

Data obtained for shoots (Table 3, 4) indicated metal accumulation lower than those measured in roots of both Paulownia hybrid lines. The results showed a more pronounced root accumulation of the toxic element Pb and the essential metal Zn

Table 3. Metal contents in P. tomentosa x fortunei line TF 01 exposed 10 days to different concentra-tions of Cd, Pb and Zn and bioaccumulation coefficients. Data are presented as means of five replica-tions ± standard error. Different letters indicate significant differences assessed by Fisher’s LSD test

(P ≤ 0.05) after performing of one-way ANOVA analysis.

Treatments(mg L-1)

Metal content (mg kg DM -1)ВС

Leaf Stem Shoots RootsCd0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -0.5 1.8±0.2ab 1.1±0.1ab 2.9±0.2a 26.3±2.4a 58.42.5 3.9±0.4ab 10.1±1.4ab 14.0±0.4a 541.7±56.8b 222.35.0 11.9±1.3abc 9.5±0.9ab 21.4±1.3a 458.2±32.7b 95.9Pb0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -5.0 5.6±0.6abc 7.6±0.8ab 13.2±0.4a 1580.8±231.5c 318.810.0 19.6±2.8bc 11.6±1.9ab 31.2±2.7a 2608.0±341.7d 263.920.0 24.0±2.5c 14.4±3.8b 38.4±3.5a 7941.2±563.8e 398.9Zn0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -10.0 177.6±12.8d 141.6±10.5c 319.2±12.8b 488.0±34.2b 80.720.0 274.8±23.5f 201.2±15.9d 476.0±24.5c 843.2±67.3b 65.930.0 240.0±23.4e 221.6±12.8e 461.6±65.9c 1566.8±123.5c 67.6

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Table 4. Metal contents in P. elongata x fortunei line EF 02 exposed 10 days to different concentra-tions of Cd, Pb and Zn and bioaccumulation coefficients. Data are presented as means of five replica-tions ± standard error. Different letters indicate significant differences assessed by Fisher’s LSD test

(P ≤ 0.05) after performing of one-way ANOVA analysis.

Treatments (mg L-1)

Metal content (mg kg DM -1)ВС

Leaf Stem Shoots RootsCd0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -0.5 0.3±0.02a 0.5±0.03a 0.8±0.02a 55.0±3.4a 113.32.5 0.9±0.04a 3.1±0.04a 4.0±0.24ab 146.5±26.8b 60.25.0 2.6±0.03a 12.0±0.91a 14.6±1.30b 551.8±52.7e 111.6Pb0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -5.0 1.2±0.61a 2.4±0.03a 3.6±1.40ab 540.0±23.1e 108.710.0 2.4±0.08a 9.6±0.09a 12.0±0.70ab 1592.0±34.2g 160.420.0 6.4±0.05a 4.8±0.04a 11.2±0.05ab 4220.8±56.3h 211.6Zn0 0.0±0.0a 0.0±0.0a 0.0±0.0a 0.0±0.0a -10.0 92.1±2.8b 112.4±10.5b 204.5±12.8c 234.5±31.2c 43.920.0 173.4±21.5c 155.6±15.9c 329.0±14.5d 457.8±47.3d 39.330.0 177.4±23.4c 243.6±12.8d 421.0±15.9e 622.8±56.5f 34.8

compared to Cd. The accumulation of Pb, Cd and Zn in whole plants during the hy-droponic experiments of the present study increased in the order of Pb < Zn < Cd and the BC increased in the order of Pb < Cd < Zn. The BC of three metals in two popu-lations of Chromolaena odorata (L.) King, Robinson grown in similar hydroponic conditions increased in the same order of Pb < Cd < Zn, but the BC values of Pb and Cd were greater than 1000. These results confirm C. odorata as hyper-accumulator which has a potential for phytoremediation of metal contaminated soils (Tanhan et al., 2007). Our results obtained were lower than that of C. odorata. Sedum alfredii Hance is proposed as a new Zn/Cd hyper-accumulator and Pb accumulator (Yang et al., 2002, 2004; Xiong et al., 2004 a, b). Under hydroponic culture, Zn concentra-tion and accumulation in shoots reached 19.67 g kg -1 at Zn supplying level 80 mg L-1. The amount of Cd accumulated in shoots reached 9000 and 6500 mg kg-1 at Cd level of 200 and 400 μM L-1 (Yang et al., 2004) and 11 000 mg kg-1 at 600 μM L-1 Cd (Zhou, Qiu, 2005). Xiong et al. (2004 a, b) found that the Pb contents in shoot of S. alfredii are 0.17 and 1.17 g kg -1 at 200 and 1500 μM L-1. Our results showed that two Paulownia hybrid lines possessed limited metal accumulation potential as compared to herbaceous hyper-accumulators in hydroponic conditions tested. The removal of heavy metals from the nutrient solution was due to action of phytostabi-

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Fig. 2. Stomatal conductance (gs) in the mature leaves of P. tomentosa x fortunei line TF 01 and P. elongata x fortunei line EF 02, grown in hydroponic and exposed 10 days to different concentra-

tions of Cd (A), Pb (B) and Zn (C).

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Fig. 3. Transpiration rate (E) in the mature leaves of P. tomentosa x fortunei line TF 01 and P. elongata x fortunei line EF 02, grown in hydroponic and exposed 10 days to different

concentrations of Cd (A), Pb (B) and Zn (C).

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Fig. 4. WUE in the mature leaves of P. tomentosa x fortunei line TF 01 and P. elongata x fortunei line EF 02, grown in hydroponic and exposed 10 days to different concentrations of Cd (A), Pb (B)

and Zn (C).

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lization mechanism rather than phytoextraction mechanism. Several studies reported different results on accumulation and translocation patterns even in the same plant species. The accumulation pattern in roots of Brassica juncea plants grown under hydroponics increased in the order of Cd < Pb < Cu < Zn whereas the BC is Zn < Cd < Cu < Pb (Dushenkov et al., 1995). Shoots of B. juncea grown in a fertilized sand:perlite mixture showed an accumulation pattern Cu < Cd < Pb < Zn whereas the phytoextraction coefficients increased in the order of Pb < Cu < Zn < Cd (Nanda-Kumar et al., 1995). This discrepancy arises due to variation in heavy metal concen-tration, form of metal present, and plant species (Kim et al., 2003). The metals have a different mobility and they were transported from roots to shoots to different manner. Cd and Zn were more mobile than Cu and Pb (Greger, 2004). Zn was translocated extensively as it was essential to the plant metalloenzymes (Delhaize et al., 1985; van Assche, Clijsters, 1990) and photosynthesis (Hsu, Lee, 1988), while Pb and Cd were toxic to plants. Maybe Zn was transported from roots to shoots to the form of chelates with organic acids while Pb and Cd were translocated as divalent cations (Mench et al., 1988).

Gas exchange measurements are often useful to detect the more sensitive site of action of heavy metals. In Zea mays L. simultaneous inhibition of photosynthe-sis and transpiration by Cd (Carlson et al., 1975) suggested that the primary effect should be on stomatal function and as a result decreased the water use efficiency. Our results showed that gs and E decreased sharply with increasing of metal concentra-tions in solution, but PN is enhanced, namely in the leaves of TF 01 plants (Fig. 2, 3, 1). Hermle et al. (2007) found that seedlings of Salix viminalis responded to heavy metal stress with increasing photosynthetic rate without growth reduction. Net CO2 assimilation and stomatal conductance were significantly reduced and ultramorpho-logical and physiological modifications were induced in P. tomentosa after treatment with 2000 µM Zn added in nutrient solution (Azzarello et al., 2012). Our results showed that PN was enhanced in the leaves of TF 01 plants at lower level of Zn in the nutrient solution. Effective mechanism of Zn sequestration and accumulation in peti-ole cell walls and root hairs was showed in Paulownia species at highest Zn levels.

Higher WUE in the variants with slight and moderate metal concentrations can be interpreted as an attempt of plants to improve their water regime. Despite of highest dry biomass investment in roots in comparison with aerial part under in-creased concentrations of heavy metals in nutrient solution it is possible that reduced water uptake by damaged roots reflected in higher WUE. High WUE is largely a function of reduced water use rather then a net improvement in plant production or biochemistry of assimilation. WUE commonly achieved via moderated growth, reduced leaf area or short growth duration. Barcelò, Poschenrieder (1990) reported that the disturbed water relations to plants comprised one of the main reasons for the heavy metal phytotoxicity. Heavy metal contamination limited root growth (limiting water uptake) and increased the degree of root suberization and lignification (in-creased resistance to water flow) as well as leaf senescence and abscission.

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CONCLUSION

It is concluded that 58th old Paulownia hybrid lines grown under hydropon-ic-culture conditions possessed a high potential for heavy metal accumulation in the roots. The strong biomass and leaf areas reduction, but increased root/shoot dry biomass are observed after treatment with different concentrations of heavy met-als. Total leaf area/total dry biomass ratios increased slightly after treatment with highest concentrations of Cd, Pb and Zn. Net photosynthetic rate (PN) and water use efficiency (WUE) are enhanced namely in the leaves of TF 01, but stomatal conduc-tance (gs) and transpiration rate (E) decreased sharply in both lines with the increase in metal concentrations in solution. The results showed that Paulownia tomentosa x fortunei line TF 01 is more tolerant to action of heavy metals than Paulownia elon-gata x fortunei line EF 02. Because the bioaccumulation coefficients (BC) for Pb and Cd are greater than 100 in both lines they can successfully as accumulators for phytoremediation of soils contaminated with these metals.

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