Arbuscular mycorrhizal fungi differentially affect the ...

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Environmental Pollution 153 (2008) 137e147 www.elsevier.com/locate/envpol

Arbuscular mycorrhizal fungi differentially affect the response to high zinc concentrations of two registered poplar clones Guido Lingua a,*, Cinzia Franchin b, Valeria Todeschini a, Stefano Castiglione c,1, Stefania Biondi b, Bruno Burlando a, Valerio Parravicini c, Patrizia Torrigiani b, Graziella Berta a a

Dipartimento di Scienze dell’Ambiente e della Vita, Universita` del Piemonte Orientale ‘‘Amedeo Avogadro’’, Via Bellini 25/G, I-15100 Alessandria, Italy b Dipartimento di Biologia evoluzionistica sperimentale, Universita` di Bologna, Via Irnerio 42, I-40126 Bologna, Italy c Dipartimento di Biologia, Universita` di Milano, Via Celoria 25, I-20100 Milano, Italy Received 19 February 2007; received in revised form 6 July 2007; accepted 17 July 2007

Inoculation with arbuscular mycorrhizal fungi can improve poplar tolerance to heavy metals in phytoremediation programmes. Abstract The effects of a high concentration of zinc on two registered clones of poplar (Populus alba Villafranca and Populus nigra Jean Pourtet), inoculated or not with two arbuscular mycorrhizal fungi (Glomus mosseae or Glomus intraradices) before transplanting them into polluted soil, were investigated, with special regard to the extent of root colonization by the fungi, plant growth, metal accumulation in the different plant organs, and leaf polyamine concentration. Zinc accumulation was lower in Jean Pourtet than in Villafranca poplars, and it was mainly translocated to the leaves; the metal inhibited mycorrhizal colonization, compromised plant growth, and, in Villafranca, altered the putrescine profile in the leaves. Most of these effects were reversed or reduced in plants pre-inoculated with G. mosseae. Results indicate that poplars are suitable for phytoremediation purposes, confirming that mycorrhizal fungi can be useful for phytoremediation, and underscore the importance of appropriate combinations of plant genotypes and fungal symbionts. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Arbuscular mycorrhizae; Phytoremediation; Polyamines; Poplar; Zinc

1. Introduction Abbreviations: AMF, arbuscular mycorrhizal fungi; DW, dry weight; NoMet, plants grown in soil unsupplemented with zinc; PA(s), polyamine(s); Put, putrescine; Spd, spermidine; Spm, spermine; TFL, translocation factor from roots to leaves; TFS, translocation factor from roots to shoots; Zn, plants grown in soil supplemented with zinc; ZnGi, plants inoculated with G. intraradices and grown in soil supplemented with zinc; ZnGm, plants inoculated with G. mosseae and grown in soil supplemented with zinc. * Corresponding author. Tel.: þ39 0 131 360 233; fax: þ39 0 131 360 390. E-mail address: [email protected] (G. Lingua). 1 Present address: Dipartimento di Chimica, Universita` di Salerno, Stecca 7, Via Ponte don Melillo, I-84084 Fisciano (SA), Italy. 0269-7491/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2007.07.012

Phytoremediation is an emerging branch of science and technology, which uses plants for this purpose. It is of special interest for those pollutants, such as metals, that can be immobilized (phytostabilization) or taken up by the roots and transferred to above-ground organs of the plant (phytoextraction). It is an environment-friendly approach and has reduced costs when compared to traditional physicalechemical methods (Faison, 2004). In addition, only phytoremediation permits the restoration of the microbial soil community, an important aspect from an ecological point of view.

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Plants used for phytoremediation should be fast-growing, have a wide-spreading root system and large biomass, should be easy to propagate, and able to store large amounts of the metal of interest (Kramer and Chardonay, 2001). Although most true metal hyper-accumulators have been found among herbaceous plants (Macek et al., 2004), some trees, such as poplar and willow, possess several of the features mentioned above. Amongst these, the incomparably larger biomass make their use very favourable, in spite of a reduced efficiency of accumulation (Pulford and Watson, 2003). The use of poplars has been proposed for the phytoremediation of various pollutants, including hydrocarbons, herbicides, and metals (Di Baccio et al., 2003). The natural ability of plants in the removal of contaminants can be integrated and improved by arbuscular mycorrhizal fungi (AMF), which are naturally present in the roots of most plant species where they form a mutualistic association (Smith and Read, 1997). Besides improving plant mineral nutrition and health, they are themselves involved in the uptake and detoxification of various pollutants, including heavy metals (Turnau et al., 2005). The mycobiont thereby enhances plant growth as well as tolerance towards biotic (Lingua et al., 2002; Berta et al., 2005) and abiotic stresses (Leyval et al., 1997; Volante et al., 2005). In some cases, increased tolerance towards heavy metals observed in mycorrhizal plants has been explained by reduced metal translocation to the above-ground organs of the plant (Schutzendubel and Polle, 2002). However, different fungaleplant associations can provide different responses, and, therefore, further information is required to understand whether mycorrhizal plants, and specific fungal taxa or strains, enhance the bio-accumulation or bio-stabilization of pollutants and through what mechanisms. Zinc is an essential micronutrient for plants, but when present in excess it can produce noxious effects, similar to those due to toxic, non-essential elements, like cadmium or lead. Macroscopic effects concern plant growth (Rout and Das, 2003), especially root development, via profound alterations of mitotic activity and cell expansion (Eun et al., 2000; Liu et al., 2003) and via genotoxic damage (Borboa and de la Torre, 1996). The mechanisms of zinc toxicity are extremely diverse and they take place through various biochemical processes. Amongst these, there is the interaction with various functional groups of proteins, mainly with SH groups, resulting in the alteration of the reactive centre of many enzymes; in addition, the reduction of photosynthetic rate and chlorophyll content (Di Cagno et al., 1999), alterations in membrane permeability (Herna´ndez and Cooke, 1997), oxidative damage (Briat and Lebrun, 1999), and increases in the cell polyamine pool (Sharma and Dietz, 2006) have been described. Polyamines (PAs) are low molecular-weight aliphatic amines that are present in all living organisms. In higher plants, the main PAs, spermidine (Spd) and spermine (Spm) and their diamine precursor putrescine (Put), are essential for growth and development, by stabilizing nucleic acids and favouring transcription and translation (Bagni et al., 1993). PAs can be conjugated to hydroxycinnamic acid derivatives to produce hydroxycinnamoylamides (also known as soluble

conjugated PAs), or to high molecular-mass compounds, such as cell wall components (insoluble conjugated PAs), which can be regarded as defense- or stress-related compound (Flores and Martin-Tanguy, 1991). Up-regulation of PA metabolism has been reported in response to several environmental stress conditions (Urano et al., 2003), including heavy metals in a variety of herbaceous plant species (Pirintsos et al., 2004; Scoccianti et al., 2006). In in vitro-grown microcuttings of Populus alba Villafranca, a concentration-dependent zinc-induced accumulation of free and conjugated Put and Spd has been reported (Franchin et al., 2007). This study is part of a broader project on the selection of poplar clones suitable for phytoremediation purposes, and it complements field investigations on a contaminated site in which the main metal pollutants are copper and zinc (Lingua et al., 2005), as well as in vitro (Castiglione et al., 2007; Franchin et al., 2007) or other greenhouse (Todeschini et al., 2007) experiments carried out in parallel. In the present paper, the effect of treatments with a high concentration of zinc on two registered clones of poplar, inoculated or not with two AMF, Glomus mosseae or Glomus intraradices, was investigated. Special attention was paid to: (i) the extent of colonization by AMF; (ii) plant growth; (iii) metal accumulation in the different plant organs; and (iv) leaf PA concentration.

2. Materials and methods 2.1. Biological material Two registered poplar clones, Villafranca (P. alba L.) and Jean Pourtet (Populus nigra L.), provided by the C.R.A. e Istituto di Sperimentazione per la Pioppicoltura (Casale Monferrato, AL, Italy) as 20-cm-long cuttings, were used for all the experiments. They were collected in February 2003 and stored at 4  C until use. Inocula of the two AMF, Glomus mosseae (Gerd. and Nicol.) Gerdemann and Trappe BEG 12, and G. intraradices (Schenck and Smith) BB-E, were supplied by Biorize (Dijon, France) on a sand carrier; they contained a minimum of 60,000 propagules kg1. Neither of the fungal species originated from contaminated soil.

2.2. Plant growth and fungal inoculation The cuttings were removed from cold storage and placed overnight under running tap water. They were then put into 20-cm-high plastic pots (750 mL), containing a 3:1 mixture of heat sterilized (160  C, 4 h) quartz sand: autoclaved soil (two 1-h treatments under flowing steam). Average sand particle diameter was 3e4 mm; soil came from an agricultural area (Tortona, AL, Italy), it was a sandy loam soil (according to USDA), and it had the following chemical features: organic matter ¼ 2.24% DW; N < 0.01% DW; K ¼ 0.0237% DW; P ¼ 0.0026% DW; pH ¼ 6.21; and Zn ¼ 47.5 mg kg1 DW. The chemical analyses (by Idrocons s.r.l., Rivalta Scrivia, Tortona, Italy) were carried out by ICPOES, as described below (Section 2.6). Eight cuttings of each clone were inoculated separately with one of the two AMF by adding 25% (v/v) inoculum to the substrate (pre-inoculated samples). Eight cuttings per clone were kept axenic (non-inoculated controls). The plants, irrigated three times a week with tap water, were maintained in a growth chamber (16-h photoperiod, t ¼ 24  C, 150 mE m2 s1 irradiance at pot height) for 40 days, at the end of which four pre-inoculated plants of each clone per treatment (see below) were harvested to evaluate the extent of colonization in newly formed adventitious roots.

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2.3. Metal treatments The remaining 40-day-old plants (pre-inoculated and non-inoculated) were transplanted into 25-L plastic pots containing non-sterile soil (from the same batch used before), and transferred into a glasshouse. Part of the soil used for the preparation of the pots was supplemented with ZnCl2 (equivalent to 300 mg kg1 zinc), and the other part was not supplemented, resulting in four different treatments (four plants each): (i) NoMet, plants grown in soil unsupplemented with zinc; (ii) Zn, plants grown in soil supplemented with zinc; (iii) ZnGm, plants pre-inoculated with G. mosseae and grown in soil supplemented with zinc; and (iv) ZnGi, plants pre-inoculated with G. intraradices and grown in soil supplemented with zinc. Since the experiment was a simulation of a field trial, soil was not sterilized (i.e., it contained naturally occurring propagules of AMF), so that spontaneous mycorrhization could be evaluated in the presence or absence of added zinc. After about 6 months, the plants were harvested and analyzed for a series of parameters, as detailed below. Leaves used for the analysis of PAs (frozen in liquid nitrogen and stored at 80  C until use) and metal content (dried at 70  C for 72 h) were of comparable size and position on the plant.

2.4. Analysis of growth parameters and mycorrhizal colonization

139

Translocation factors (TFs) for zinc were calculated for the stems (TFS) and leaves (TFL) as the ratio between the metal concentration in the stem (or in the leaves) and the metal concentration in the roots. Total metal content per plant was calculated from the average weight of each organ multiplied by the relative average zinc concentration.

2.7. Correlation between Put and zinc concentrations In order to evaluate the correlation between zinc and Put concentrations, three samples of leaves from each zinc treatment (pre-inoculated or not with AMF) were divided into two sub-samples, and analyzed separately for zinc or Put concentration, as previously described. Results were then plotted as the soluble conjugated to free Put ratio versus zinc concentration. A linear regression and the corresponding R2 were calculated.

2.8. Statistical analyses Mean values and standard errors were calculated, and the data compared by analysis of variance (ANOVA) and the F post hoc test, with p < 0.05 as the significance cut-off.

3. Results Various morphological parameters were recorded: plant height; fresh and dry weight of leaves, shoots and roots; leaf number and surface area. Leaf area was measured by means of a scanner and of a specific software package (WinRhizo, Regent Instruments, Quebec, Canada). At the end of the experiments, the degree of mycorrhizal colonization of all plants, pre-inoculated or not, was evaluated microscopically, according to Trouvelot et al. (1986). Briefly, at least 30 1-cm-long root segments were sampled from each root system, cleared in 10% KOH at 45  C for 1 h, washed twice in 10% KOH for 24 h, and stained with 1% methyl blue in lactic acid (Lingua et al., 1999), and mounted on slides. Microscopic observations were carried out at 50e 630 magnifications. Results are expressed as intensity of colonization, i.e., percentage of colonized roots (M%), and abundance of arbuscules in the colonized portion of the root (a%).

2.5. HPLC analysis of polyamine content Plant material (frozen leaves) was homogenized with 10 vol. of 4% perchloric acid (PCA), kept on ice for 45 min, and centrifuged at 15,000g for 30 min. Aliquots of the supernatant were derivatized with dansylchloride in order to determine free PAs. The remaining part of the supernatants and the pellets resuspended in 4% PCA were hydrolysed with 12 N HCl at 110  C overnight in order to release PAs from the PCA-soluble and -insoluble conjugated forms, respectively. After evaporating the HCl and resuspending the dry residue in 4% PCA, hydrolysed samples were also dansylated. Put, Spd and Spm were separated and identified by HPLC as described by Franchin et al. (2007).

2.6. Chemical analyses Approximately 0.5 g DW was used for the determination of zinc concentration in leaves, stems and roots, separately. Samples were weighed and then digested in 10 mL concentrated HNO3 in a CEM MARS 5 microwave digestor (Cologno al Serio, BG, Italy). The digested material was filtered on 45-mm filters, and then deionized water was added to a final volume of 100 mL. Metal concentration was assessed by means of a calibration curve, after measurement by Inductively Coupled Plasma Optic Emission Spectrometry (ICP-OES) using an IRIS Advantage ICAP DUO HR series (Thermo Jarrell Ash, Franklin, MA, USA) spectrometer. The same method was used for the determination of phosphorus concentration in leaves, and of N, P, K and zinc in soil. Certified standards (BCR 062, 100, 129 and 145R, by the Institute for Reference Materials and Measurements, Ratieseweg, Belgium), with known element concentration, were analyzed with the samples in order to confirm the correctness of the procedure.

3.1. Mycorrhizal colonization In Villafranca, AMF colonization occurred although it never reached very high levels. At the time of transplanting, M% was zero in NoMet plants, while in pre-inoculated plants it was about 3%. At the end of the experiment, M% was never over 16% (Fig. 1a). Zinc caused the total inhibition of spontaneously occurring M% (Fig. 1a). However, pre-inoculation with either G. mosseae or G. intraradices allowed zinc-treated plants to achieve a colonization level comparable to that of control plants grown without the metal (Fig. 1a). The abundance of arbuscules, however, was severely reduced by zinc treatment (Fig. 1a). In Jean Pourtet, colonization by AMF was always more extensive than in Villafranca. At the time of transplanting, M% was zero in uninoculated (NoMet) plants, while it was between 3 and 4% in pre-inoculated plants. Zinc had no significant effect on spontaneously occurring mycorrhization in this clone (Fig. 1b), while pre-inoculation resulted in levels of AMF colonization that were even higher than in NoMet plants (Fig. 1b). Arbuscule abundance was lowest in Zn plants, while in preinoculated ones, a% was not affected by the presence of the metal (Fig. 1b). No signs of ectomycorrhizal colonization were observed in any of the treatments and in either of the clones. 3.2. Plant growth parameters In Villafranca and Jean Pourtet control plants, growth during the considered time period was highly comparable in terms of shoot length, shoot weight and root growth (Fig. 2). However, the effects of high zinc concentration on biomass production differed in the two clones. In Villafranca, zinc treatment affected all the growth parameters: in particular, shoot growth was inhibited by 50% and root growth by 70%. Pre-inoculation with G. mosseae

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Fig. 1. Mycorrhizal colonization (M%, white columns) and arbuscule abundance in the colonized area (a%, black columns) in the root system of P. alba Villafranca (a) or of P. nigra Jean Pourtet (b). NoMet: plants not supplemented with metal; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05).

allowed the plants to grow as the NoMet ones also in the presence of zinc, with the exception of roots whose development remained severely compromised. No beneficial effects were observed with G. intraradices pre-inoculation of zinc-treated plants (Fig. 2aed). In Jean Pourtet, the addition of zinc reduced, in comparison to NoMet plants, root biomass, but only shoot length as far as the aerial parts of the plant are concerned (Fig. 2eeh). This growth reduction was never alleviated by inoculation with AMF. 3.3. Zinc translocation and accumulation in different organs In all the organs of Villafranca control plants, the concentration of zinc was always below 50 mg kg1 DW. Metal concentration significantly increased in all the organs when the plants were grown on zinc-supplemented soil, and was always higher in planta than in the soil (Fig. 3aec). The highest metal concentration was observed in the leaves, while the concentration in the stems and in the roots was significantly lower. G. mosseae slightly, but significantly, reduced zinc accumulation only in the leaves, in comparison with Zn plants, while G. intraradices never affected zinc levels in any organ (Fig. 3). However, as shown in Table 1, the highest total content of

zinc was found in ZnGm plants (24.26 mg per plant on average, of which about 60% in the leaves), followed by Zn and ZnGi ones, while in NoMet plants it was one order of magnitude lower. In Villafranca NoMet plants, TFL was approximately 1, and about 5 in all zinc-treated ones (Table 1). However, TFL was significantly lower in ZnGm plants than in Zn ones. On the contrary, TFS, which was much lower than TFL in all cases, showed no significant differences among treatments (Table 1). The TFL for zinc was, in all cases, higher for Villafranca than for Jean Pourtet. In Jean Pourtet plants, metal concentration significantly increased in all the plant organs when the plants were grown on zinc-supplemented soil (Fig. 3eeg). Again, the highest concentration was observed in the leaves, while roots and stems exhibited similar concentrations. Mycorrhizal inoculation significantly reduced metal accumulation in zinc-treated plants only in the stems (Fig. 3f), but not in the other organs. The total zinc content was highest in Zn plants (21.09 mg per plant), followed by ZnGi, ZnGm and control plants, and values were generally lower than in Villafranca (Table 1). As shown in Table 1, both translocation factors were higher, up to about 5-fold for TFL, in zinc-treated plants of the P. nigra clone than in control ones. Different from Villafranca, TFL was not affected by AMF, and TFS was about 20e30% lower in mycorrhizal plants than in Zn ones. 3.4. Leaf phosphorus concentration In Villafranca, the lowest phosphorus concentration was recorded in NoMet plants and this value was significantly different from that of the other three treatments, which were not significantly different between themselves (Table 1). By contrast, in Jean Pourtet, the highest phosphorus concentration was found in ZnGm leaves, and it was significantly different from the other three treatments, which were not significantly different between themselves (Table 1). 3.5. Leaf polyamine profile Both free, and soluble and insoluble conjugated Put and Spd were detected in NoMet Villafranca plants, while the small amount of Spm present only occurred in the soluble conjugated form (Fig. 4aec). Soluble conjugated PAs were the most abundant (about 3-fold relative to the free form for Put and Spd); insoluble conjugated ones were detected in low amounts and were never affected by zinc in the presence or in the absence of AMF (data not shown). The addition of zinc in the soil caused a 3-fold increase in the amount of free Put, which reverted the reciprocal ratio between the free and soluble conjugated forms compared with NoMet plants (Fig. 4a). Spm levels were also affected in Zn plants: free Spm was present, though at low concentration, at the expense of the soluble conjugated form that significantly decreased compared with NoMet controls (Fig. 4c). No changes in Spd titers were detected (Fig. 4b). In ZnGm Villafranca plants, the PA pattern did not differ from that of NoMet controls with the only exception of the

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Fig. 2. Shoot length (cm), epigeous and root biomass (g), leaf area (cm ) of P. alba Villafranca (aed) and of P. nigra Jean Pourtet (eeh). NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05). Notice that the different graphs have different scales.

presence of some free Spm (Fig. 4c). Instead, in the presence of G. intraradices, zinc-supplemented plants (ZnGi) exhibited a different PA pattern relative to NoMet plants. In fact, while free Put and Spd concentrations were not affected, the levels of their soluble conjugated forms significantly decreased (2e3 times); conjugated Spm was also decreased, while a small amount of the free one persisted. In the leaves of zinc-treated plants of Villafranca, there was a reverse correlation between zinc concentration and the soluble conjugated to free Put ratio (Fig. 5). In Jean Pourtet NoMet plants, overall PA titres were lower than in Villafranca, and only Put was more abundant in the soluble conjugated than in the free form (Fig. 4). In the presence

of zinc, the PA pattern was not significantly altered (Fig. 4def). In ZnGm and ZnGi plants, the only change induced relative to Zn plants was an approximately 2-fold increase in soluble conjugated Put concentration (Fig. 4d). 4. Discussion The present study shows that inoculation with AMF can alleviate the growth and metabolic perturbation/stress induced by a high concentration of zinc in poplar under greenhouse conditions, and that the response depends upon both the fungal and plant species.

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Fig. 3. Zinc concentrations (mg kg DW) in the different organs of P. alba Villafranca (aec) and of P. nigra Jean Pourtet (eeg) plants, treated or not with zinc and pre-inoculated or not with G. mosseae or G. intraradices. NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05). Notice that the different graphs have different scales.

4.1. Zinc inhibits spontaneous root colonization by AMF Plants grown on non-sterile soil enabled us to evaluate the effects of zinc contamination on spontaneous root colonization by naturally occurring AMF. Growth on non-sterile soil is

useful for simulating conditions similar to those met in the field, and to assess the consequences of AMF colonization (Orlowska et al., 2005). Roots of NoMet plants were spontaneously colonized by AMF, while no traces of ectomycorrhizal colonization were

Table 1 Total zinc content per plant in Villafranca and Jean Pourtet following the various treatments, calculated from the average weight of each organ multiplied by the relative average zinc concentration; zinc translocation factors, relative to stems (TFS) and leaves (TFL), calculated as the ratio between zinc concentration in stems (or leaves) and zinc concentration in roots  standard errors; P concentration in leaves  standard errors Treatment

Total Zn content (mg plant1)

TFS

P concentration (g kg1 DW)

Villafranca

NoMet Zn ZnGm ZnGi

1.78 18.35 24.26 17.63

0.615  0.125 0.906  0.063 0.757  0.194 0.790  0.150

a a a a

1.183  0.294 5.785  0.540 4.378  0.477 5.170  0.303

a b c bc

1.000  0.009 1.581  0.118 1.213  0.042 1.158  0.069

a b b b

Jean Pourtet

NoMet Zn ZnGm ZnGi

1.17 21.90 12.67 14.51

0.581  0.092 1.232  0.125 0.844  0.038 0.957  0.046

a b c c

0.794  0.107 3.937  0.243 4.124  0.165 4.279  0.140

a b b b

1.567  0.010 1.481  0.021 1.735  0.078 1.384  0.022

a a b a

TFL

NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with Glomus mosseae; ZnGi: plants treated with zinc and pre-inoculated with Glomus intraradices. Different letters indicate significant differences, along the columns ( p < 0.05).

G. Lingua et al. / Environmental Pollution 153 (2008) 137e147

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Fig. 4. Mean concentration and standard error (bars) of free (white columns) and soluble conjugated (black columns) polyamines in leaves of P. alba Villafranca (aec) and of P. nigra Jean Pourtet (def), treated or not with zinc, and pre-inoculated or not with G. mosseae or G. intraradices. NoMet: plants not supplemented with zinc; Zn: plants supplemented with zinc; ZnGm: plants treated with zinc and pre-inoculated with G. mosseae; ZnGi: plants treated with zinc and pre-inoculated with G. intraradices. Bars represent standard errors. Different letters indicate significant differences ( p < 0.05).

observed. Such results are in agreement with previous data reporting that AMF colonization is prevalent in young poplar plants, while ectomycorrhizal colonization is dominant in older ones (Aguillon and Garbaye, 1989; Khasa et al., 2002). Consistent with the agricultural origin of the soil

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Leaf [Zn] (mg kg-1 DW) Fig. 5. Correlation between zinc concentration and the soluble conjugated to free putrescine ratio (conj Put/free Put) in leaves of P. alba Villafranca plants treated with zinc, and pre-inoculated or not with AMF.

(rich in phosphate), colonization was relatively low, as M% was always below 20% in both poplar clones, with higher rates for Jean Pourtet in comparison with Villafranca, despite similar arbuscule abundance. In zinc-treated (Zn) plants of both clones, M% was close to zero. The negative effect of zinc on fungal spore production has previously been reported (Del Val et al., 1999). However, in the present case, colonization of non-inoculated plants also relied on propagules already present in the soil. Therefore, zinc, besides reducing spore production, may have prevented root colonization by some other action, either on the fungus (spore vitality, germination, hyphal elongation, or appressoria formation) or on the host plant, for example, by triggering defence responses, modifying hormonal balances, or altering root exudate composition, the latter an important factor for hyphal branching and root colonization by AMF (Akiyama et al., 2002, 2005). By contrast, root colonization was not hampered by the presence of the metal in the soil when the plants were pre-inoculated with AMF; however, a% was close to zero in ZnGm Villafranca, but not so in Jean Pourtet. Different levels of AMF

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colonization, depending upon plant genotype/species, were previously described in poplars growing on heavy metal-polluted soil (Taka`cs et al., 2005). Variations also seem to depend on the metal element, since, as reported by Rivera-Becerril et al. (2002), cadmium did not affect the levels of colonization in three different genotypes of pea. 4.2. Zinc impairs growth, and AMF can, in some cases, alleviate this effect Present results indicate that toxic levels of zinc were reached in both poplar clones, though to a different extent. Thus, while in non-inoculated Villafranca plants zinc dramatically reduced the values of all the growth parameters analyzed, in Jean Pourtet the effects were less severe as they involved only shoot length and root biomass. The amount of zinc accumulated in leaves far exceeded even the highest values reported by Laureysens et al. (2004) for senescing leaves in a poplar coppice culture growing on a ‘‘slightly contaminated soil’’, and greatly outranged the ‘‘toxic levels’’ defined by Kabata Pendias and Pendias (1984). Growth reduction is, in fact, one of the prominent responses to metal stress (Adriaensen et al., 2003; Taka`cs et al., 2005), even though some authors (Di Baccio et al., 2003; Vandecasteele et al., 2005) have reported that biomass was not affected by zinc treatments. Within a certain range (up to about 800 mg kg1) Laureysens et al. (2004) found no negative correlation between biomass production and leaf zinc concentration in poplars. Different growth conditions, or soil and site characteristics, may account for such discrepancies; different age and physiological status of the plant, and/or species or clonal sensitivity are also likely factors (see Villafranca versus Jean Pourtet in this study). In Villafranca plants pre-inoculated with AMF, the negative effects on growth exerted by zinc were alleviated. Alleviation by AMF of heavy metal-induced stress was reported in other mycorrhizal systems, including Pisum sativum/G. intraradices (Rivera-Becerril et al., 2002). More recently, Janouskova et al. (2005) reported that cadmium concentrations were lower in the biomass of mycorrhizal tobacco plants than that of nonmycorrhizal ones, suggesting that G. intraradices decreased cadmium uptake or its translocation to the shoots. In general, however, the mechanisms involved in conferring increased tolerance/decreased toxicity have not been clarified as yet and may be quite diverse. For example, in analogy with the beneficial effects exerted by ectomycorrhizas, improved plant nutrition, accumulation of the metal in microbial structures, and/or chelation of the metal by fungal exudates may represent some of the mechanisms involved (Leyval et al., 1997; Jentschke and Godbold, 2000). In the present case, because of the very low arbuscular abundance and because of the lack of differences in phosphorus concentration between Zn and ZnGm Villafranca plants, it is unlikely that plants benefited from improved nutrition. Metal effects on growth were much less pronounced in Jean Pourtet, and mycorrhizal inoculation induced few modifications, in spite of the fact that AMF more extensively colonized this clone than Villafranca. Actually, in Jean Pourtet, metal concentration was much lower (in leaves 40% less than in

Villafranca) and phosphorus content higher than in Villafranca. It is possible that a more extensive mycorrhizal colonization and improved ability to take up phosphorus contributed to reduce zinc uptake and translocation to the leaves (Huang et al., 2000; Adriaensen et al., 2003), thereby reducing the negative effects on shoot biomass. An alternative explanation is that the presence of the fungus triggered biochemical modifications that improve the fitness of the plant, thereby increasing plant tolerance to stress. In the present work, this hypothesis is supported by the fact that, in Villafranca, changes in free Put concentration induced by zinc treatment were reverted in ZnGm plants. However, the same did not occur in Jean Pourtet. This genotypic specificity in the responses to metals and mycorrhizae confirms previous observations regarding the variable degrees of tolerance to metals within the same plant taxon (Rivera-Becerril et al., 2002; Taka`cs et al., 2005). 4.3. AMF can alter metal uptake and translocation to the different plant organs Zinc tissue content increased markedly in plants grown on zincsupplemented soil, reaching, in the leaves of Villafranca, levels up to eight times higher than in the soil. These observations are in agreement with the findings of Di Baccio et al. (2003) concerning zinc uptake and distribution in Populus  euroamericana. The idea that mycorrhizae may act by reducing metal uptake/translocation into the host plant is controversial (Marques et al., 2006). In fact, in Villafranca and Jean Pourtet poplars, the amount of zinc accumulated in mycorrhizal plants was very high, and not substantially reduced relative to noninoculated ones. The variety of responses deriving from the interaction between different fungal species and poplar clone was also evident with respect to the distribution of the metal in different organs, suggesting that mycorrhizal colonization does, in some cases, affect the mobility/allocation of metals within the plant. The high TFL observed for Zn plants confirms that poplar is a good accumulator of this metal in its leaves. In early stages of growth, as in the present case, these organs represent about 20% of the total biomass of the plant. In spite of a slight, but significant, reduction of zinc concentration in the leaves, compared with Zn plants, total zinc content in ZnGm Villafranca plants was the highest observed. At the same time, pre-inoculated plants were able to reach levels of AMF colonization comparable with those of NoMet plants. Therefore, inoculation of Villafranca cuttings with G. mosseae provided several positive results for phytoextraction purposes: plant size/biomass and AMF colonization comparable to that of NoMet plants, and higher zinc accumulation. 4.4. In Villafranca, zinc-induced changes in PAs are absent in ZnGm plants Although it is widely accepted that PA metabolism is implicated in several environmental stresses (Urano et al., 2003), studies aimed at investigating changes in endogenous PA content in plants associated with endophytes and subjected to

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some form of stress are rare. To our knowledge, this is the first time that heavy metal stress and colonization by AMF are analyzed together from the standpoint of PAs. In leaves of both poplar clones, the prevalence of conjugated PAs relative to the free form is consistent with the abundance of shikimate/phenylpropanoid-derived phenolics and hydroxycinnamate derivatives in poplar tissues; indeed, the latter can constitute up to 2e8% of leaf DW in Populus and closely related taxa (Tsai et al., 2006). The relatively low amount of free PAs in all the samples analyzed is, in any case, typical of tissues/organs exhibiting slow growth as, indeed, fully expanded leaves are (Bagni et al., 1993). In general, free Put is the most responsive PA to heavy metals, including zinc (Pirintsos et al., 2004; Scoccianti et al., 2006). In the present study, leaf zinc concentration was inversely proportional to the ratio between soluble conjugated and free Put. Hence, the metal-induced increase in free Put and the comparable decrease in the conjugated form suggest a reduction in conjugating activity. This may be detrimental for stress recovery since soluble conjugated PAs have free radical scavenger properties (Bors et al., 1989). Although some free Spm also accumulated in zinc-treated plants of Villafranca, Spd levels appeared more tightly regulated. This is in agreement with results showing that, in transgenic tobacco overexpressing S-adenosylmethionine decarboxylase, an enzyme involved in the PA biosynthetic pathway, Spd levels were not altered (Hanfrey et al., 2002). Both free and conjugated Put displayed NoMet levels in Villafranca ZnGm plants suggesting that, in the presence of this AMF, given that the amount of zinc accumulated was still very high, the toxicity of the metal was reduced/contrasted. In fact, in ZnGm plants the growth parameters of above-ground parts were also unaffected relative to controls. This is in accord with findings in Plantago lanceolata where no differences in PA content between mycorrhizal and non-mycorrhizal samples were observed when the plants exhibited similar growth (Para`di et al., 2003). Inoculation with G. intraradices was not associated with a reduction in metal translocation to the leaves, nor did it alleviate the inhibition of growth due to zinc. In fact, in ZnGi plants, the PA profile was altered in comparison with that of NoMet plants; in particular, conjugated PAs were much lower, thus supporting the contention that these compounds contrast zinc toxicity, and consistent with the observed correlation between zinc concentration and the ratio between conjugated and free Put. If the endogenous PA complement of NoMet plants is assumed to reflect a non-stress condition, then Zn and ZnGi plants were the furthest from this optimal state, while the PA profile of ZnGm plants was, instead, indicative of a long-term recovery from metal-imposed stress. In contrast to Villafranca, the PA profile remained unaltered in Zn Jean Pourtet plants relative to NoMet ones, and preinoculation with either AMF did not substantially influence this pattern. This may be associated with the fact that this clone accumulated markedly lower amounts of the metal in the leaves, that it was much more intensely colonized, and that its phosphorus content was higher. The last two features

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may be related to enhanced protection from metal stress. Taken together these results suggest that an increased endogenous PA pool is essential in order to cope with zinc-imposed stress, and that conjugates may also be needed as a way to neutralise excess phenolics produced in response to stress (Tsai et al., 2006). The most common feature of tolerance towards metals is their translocation into the leaves (Pulford and Watson, 2003), which is especially useful for phytoextraction purposes. For this tolerance mechanism to be effective it likely needs to be combined with metabolic tolerance processes occurring in that organ, such as accumulation of PAs (as shown here for Villafranca) or other protective molecules. The molecular basis for the action of PAs in alleviating stress has not been clarified, but there is evidence that they can act at several metabolic levels, as well as facilitate metal ion compartmentation (Sharma and Dietz, 2006). 5. Conclusions Both poplar clones tested here proved to be suitable for phytoremediation due to the high biomass production combined with the large amount of zinc accumulated in the leaves relative to the soil. Villafranca performed better, for phytoextraction purposes, in the presence of G. mosseae, whereas Jean Pourtet accumulated more zinc per plant in the absence of mycorrhiza without significant reduction of leaf biomass, suggesting that this clone has a different protective mechanism, possibly linked to lower zinc translocation to the leaves, more intense colonization and higher leaf phosphorus content. In conclusion, this work shows that pre-inoculation with AMF can be considered a useful strategy to ensure mycorrhizal colonization, even under conditions, such as high concentrations of heavy metals, strongly preventing the occurrence of spontaneous mycorrhization. The ability of the plant to form arbuscular mycorrhizae might be relevant for phytoremediation purposes, since this may positively affect plant stress recovery, as shown here in the case of G. mosseae-inoculated Villafranca, but not Jean Pourtet. Such differences underscore the importance of an accurate choice of plant genotype(s) for phytoremediation purposes. Acknowledgements The authors wish to thank Donata Vigani, Giuliano Bonelli, Dr. Andrea Copetta and Marco Sobrero for their valuable help throughout the experimental work, and Dr. Elisa Gamalero for critical reading of the manuscript. This work was financed by the Italian Ministry for Education, University and Research (PRIN 2003_2003077418). References Adriaensen, K., van der Lelie, D., Van Laere, A., Vangronsveld, J., Colpaert, J.V., 2003. A zinc-adapted fungus protects pines from zinc stress. New Phytologist 161, 549e555.

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