M Teresa

S D Vasconcelos

M Fernanda

C Leal

Exudates of different marine algae promote growth and mediate trace metal binding in Phaeodactylum tricornutum

Keywords: Allelopathy, Phaeodactylum tricornutum, Exudates, Organic ligands, Thiols, Trace metals

P. tricornutum was grown in filtered natural seawater enriched with nitrate, phosphate, and silicate only (control) or with exudates from itself, from Emiliania huxleyi (a coccolithophore micro-alga), Porphyra spp. (a red macro-alga) or Enteromorpha spp. (a green macro-alga).

Cathodic (and anodic) stripping voltammetry (C(A)SV) were used to determine the concentrations of trace metals, both in the medium and in the algae, as well as total Cu-complexing organic ligands in the medium and, among these, some thiols (compounds identified as cysteine-or as glutathione by CSV). Exudates of different marine micro-and macro-algae could cause allelopathic effects in P. tricornutum cultures. Cell yield of P. tricornutum was increasingly promoted by exudates of E. huxleyi > Porphyra > Enteromorpha. Although exudates strongly complex Cu (and probably other metals), their presence promoted Cu uptake. Significant changes of Ni, Cd, Fe, Zn and Mn uptake by P. tricornutum were also observed in the presence of exudates of different algal species. In addition, both intensity of production and nature of exudates released by P. tricornutum were markedly influenced by the presence of exudates of other algae, the allelopathic effects being very specific (variable from one species to another). Allelopathy will probably also occur in the aquatic environment, although to a lesser extent than in cultures, particularly during bloom events and may have effects on both chemical speciation and bioavailability of chemicals to phytoplanktonic species. Such changes might cause the predominance of some species over other species. Therefore, in future in vitro culture studies with the purpose of using them as models of the real environment, more attention should be paid to the role of algal exudates, in order to improve the environmental relevance and significance of the results.

Introduction

Any process involving secondary metabolites produced by plants, microorganisms, viruses and fungi that influences the growth and development of agricultural and biological systems excluding animals can be called allelopathy [START_REF] Romeo | Raising the beam: Moving beyond phytotoxicity[END_REF]. Although allelopathy has been mostly associated with inhibitory effects on growth, promotion of growth may also be classified as an allelopathic effect according to that broad definition. Data on allelopathy among algae are still very scarce. [START_REF] Sukenik | Inhibition of growth and photosynthesis of the dinoflagellate Peridinium gatunense by Microcystis sp. (cyanobacteria): A novel allelopathic mechanism[END_REF] have observed that Microcystis sp. (cyanobacteria) severely inhibited the growth of Peridinium gatunense (dinoflagellate) in mixed laboratory cultures. Some effects caused by the presence of different species in the same culture have been observed and attributed to the excretion of substances with allelopathic properties. Micromonas pusilla growth was inhibited in the presence of Phaerodactylum tricornutum or Heterocapsa niei marine microalgae, compared to single-species tests [START_REF] Franklin | Development of multispecies algal bioassays using flow cytometry[END_REF]. In addition to algal exudate production, competition for nutrients, including CO 2 , may have accounted for those effects. Vasconcelos et al. (2002) have studied the influence of exudates from different algal species (micro-and macro-algae) on the behaviour of Emiliana huxleyi in seawater cultures. An improvement in the final cell yield of E. huxleyi was caused by the addition of Enteromorpha spp. (a green macro-alga) exudates, and growth inhibition (a decrease in final cell yield and growth rate) was observed in the presence of P. tricornutum (a diatom) exudates. Exudates of Enteromorpha were found to be rich in thiolic compounds which were identified by Cathodic Stripping Voltammetry (CSV) as being glutathione, and P. tricornutum exudates were rich in thiolic compounds which behaved in CSV like cysteine. P. tricornutum is an efficient producer of phytochelatins (peptides that contain glutamate, cysteine and glycine with the basic formula ( -Glu-Cys) n -Gly with n = 2-11), capable of readily synthesising a wide range of phytochelatins under metal stress [START_REF] Rijstenbil | HPLC analysis of nonprotein thiols in planktonic diatoms: Pool size, redox state and response to copper and cadmium exposure[END_REF]. It has also been observed that the nature and concentration of the organic compounds present in the culture medium

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conditioned trace metal sorption, as well as the concentration and composition of the exudates produced by E. huxleyi (Vasconcelos et al., 2002).

The aim of the present work was to study the behaviour of P. tricornutum cultures in the presence of exudates produced by themselves in a previous culture, or by E. huxleyi, Porphyra spp.

(a red macro-alga) or Enteromorpha spp. Multispecies algal bioassays would be more realistic for allelopathic studies but interpretation of the results would be difficult. The study was carried out using natural coastal seawater enriched only with nitrate, phosphate, silicate and algal exudates. For the interpretation of results, concentrations of trace metals, total organic ligands, and thiol identified by CSV as cysteine and glutathione compounds were taken into consideration.

Materials and Methods

Reagents

The water used for reagent preparation and rinsing was deionised with conductivity < 0.1 µS cm -1 . The reagents and standards were prepared as before (Vasconcelos et al., 2002).

The natural seawater used in this study originated from the sea surface, 150 m from the coast at Póvoa do Varzim (Northwestern Portugal) and collected in October 2000. The seawater was pumped continuously from the ocean through a PVC pipe at a rate of 100 L s -1 , and collected at the end of the discharge pipe directly to cleaned 25 L HDPE containers. In the laboratory, the seawater (35 ‰ salinity, pH 8.0) was immediately filtered (0.1 µ m polycarbonate membrane, Millipore) and stored in 50 L HDPE containers. Seawater was sub-sampled in 500 mL HDPE bottles, stored in the dark at 4 o C and used for experiments within 1 week.

Cleaning of materials and culture media

All materials used were acid cleaned and microwave-sterilised as described before [START_REF] Leal | Copper Induced Release of Complexing Ligands Similar to Thiols by Emiliania huxleyi in Seawater Cultures[END_REF]. The culture media were sterilised by filtration (0.1 µm pore-size filter). All sample manipulations were carried out using gloves, in a Class 100 laminar flow hood in a clean room with HEPA filtered air. The absence of bacteria in the cultures was tested before and after experiments using standard bacteriological non-selective growth medium, and bacteria were not observed during the entire growth period. In addition, bacteria were not observed by using a microscope with an oil immersion lens.

Attainment of seawater enriched with exudates

Sterilised seawater was supplemented only with 176 µmol L -1 nitrate (from sodium nitrate, Merck), 7.26 µmol L -1 phosphate (from sodium dihydrogenophosphate, Merck) and 21.4 µ mol L -1 silicate (from sodium silicate, Merck, used only in the case of P. tricornutum) and was inoculated (1 L polycarbonate bottle) with 1 ml of an f/2 [START_REF] Stein | Culture Methods and Growth Measurements, Isolation and Purification (Section I)[END_REF]

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In all cases, the algae were removed by filtration through 0.45 µm cellulose nitrate Millipore filters, and rejected. The medium was characterised in terms of the concentration of total dissolved metals, total organic ligands and specific thiols which could be identified by CSV.

P. tricornutum cultures

All of the media enriched with exudates were supplemented with macronutrients as described above, sterilised, and used to grow up P. tricornutum for 10 d at 18 °C under 16 h light (and 8 h dark). For this purpose, cells from the f/2 medium (used for the stock cultures) were centrifuged (at 1500 rpm) and re-suspended in natural seawater, for elimination of EDTA and metals and then inoculated. Algae were also grown in sterilised natural seawater enriched only with the macronutrients (control media). The initial concentration P. tricornutum was approximately 1.5

x 10 6 cells L -1 .

Before and after experiments the cell numbers were counted [START_REF] Stein | Culture Methods and Growth Measurements, Isolation and Purification (Section I)[END_REF] using a microscope (Nikon, Eclipse E400).

All experiments were carried out in triplicate.

Chemical characterisation of the cultures

The total dissolved concentrations of the different trace metals ([M] d ) were determined, by CSV, in the culture media before and after algal growth, after removing the algae by filtration (0.45 µm pore-size filter) in a vacuum filtration system (Millipore) at 0.6 bar. Microscopic examination of the algae on the filter revealed that they did not suffer from rupture or breakage during filtration.

Aliquots of the media were acidified to pH 2.2 and were UV-digested (Ultramed 1000-W mercury vapour lamp, from Osram) in PTFE-capped quartz silica tubes for 1 h. Acidification at a lower pH value (pH 1.5) and/or higher UV-irradiation time resulted in statistically identical results. Metal concentrations were determined by CSV, after neutralisation of the pH with 6 M ammonia. The voltammetric equipment has been described before by [START_REF] Leal | Copper Induced Release of Complexing Ligands Similar to Thiols by Emiliania huxleyi in Seawater Cultures[END_REF]. The determination of Cu,

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Pb, Cd, Zn, Mn and Ni was carried out by CSV at pH 8.3 (0.01 M borate buffer) using, as the competitive ligand, 25 µM salicylaldoxime for Cu [START_REF] Campos | Determination of copper complexation in sea water by cathodic stripping voltammetry and ligand competition with salicylaldoxime[END_REF], 0.01 mM 8hydroxyquinoline for Pb and Cd [START_REF] Colombo | Simultaneous determination of several trace metals in seawater using cathodic stripping voltammetry with mixed ligands[END_REF], 100 µM ammonium pyrrolidinedithiocarbamate for Zn (van den [START_REF] Van Den Berg | Determination of the zinc complexing capacity in seawater by cathodic stripping voltammetry of zinc-APDC complex ions[END_REF] and 0.2 mM dimethylglyoxime for Ni [START_REF] Colombo | Simultaneous determination of several trace metals in seawater using cathodic stripping voltammetry with mixed ligands[END_REF]. The determination of Fe was carried out also by CSV at pH 6.9 (0.01 M PIPES buffer) using 0.4 mM catechol as the competitive ligand (van den Berg and Huang, 1984). The determination of Mn was carried out by CSV at pH 8.3 (0.01 M borate buffer) but without any added competitive ligand (Vasconcelos and Leal, 2001). Deposition step: E d (V) = -1.1 (Cu), -1.0 (Pb, Cd), -1.3 (Zn), -0.1 (Fe), -1.7 (Mn), -0. The concentrations of the total Cu-complexing organic ligands (C L ) in the media and the respective conditional stability constants (K' CuL ) were determined by titration of the filtered culture media with Cu and measured by CSV with ligand competition [START_REF] Campos | Determination of copper complexation in sea water by cathodic stripping voltammetry and ligand competition with salicylaldoxime[END_REF][START_REF] Leal | Copper Induced Release of Complexing Ligands Similar to Thiols by Emiliania huxleyi in Seawater Cultures[END_REF]. For CSV determinations, the pH was fixed at 8.3 (0.01 M borate) and salicylaldoxime ligand (5 µM) was added to 120 ml of sample solution. It was observed in a previous study [START_REF] Leal | Evidence for Strong Copper(I) Complexation by Organic Ligands in Seawater[END_REF]) that during the CSV Cu complexing ligand titrations (added Cu concentration in the range 0-50 nM), the detected concentration of two synthetic organic ligands (cysteine and glutathione), and the conditional stability constants of their Cu complexes, did not vary significantly when the salicylaldoxime concentration was varied in the range 2-10 µM.

Aliquots of 10 ml of sample solution were pipetted into eleven polystyrene vials also containing

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added Cu in the range of 0-200 nM (or other, if required), in approximately equal increments. The solutions were equilibrated overnight (12-15 h) prior to the determinations. The operationally labile Cu concentration ([Cu] labile ) was determined as described for [Cu] d , but without previous acidification and UV-digestion, using a 60 s adsorption step (t d ) and deposition potential (E d ) of -0.2 V, whilst stirring. This E d was used rather than the more negative used for [Cu] Thiolic compounds were identified and quantified in the culture media using a method independent of that used for C L determinations (Al-Farawati and van den Berg, 1997; [START_REF] Leal | Copper Induced Release of Complexing Ligands Similar to Thiols by Emiliania huxleyi in Seawater Cultures[END_REF]. The determinations were carried out by CSV at pH 8.3 (0.01 M borate buffer). Deposition step: E d = -0.25 V, t d = 60 s. The scanning parameters were: scan rate, 20 mVs -1 ; modulation amplitude, 25 mV; and square-wave frequency of 50 Hz. The procedure was repeated after the addition of thiol standard (cysteine or glutathione) for calibration. The cysteine peak was located at -0.47 V and the glutathione peak at -0.58 V.

Total metal concentrations in the algae ([M] algae ) (extracellular adsorption plus intracellular uptake) were determined after microwave-digestion (Milestone MLS-1200 Mega) of the filters with 1 mL of suprapure, concentrated, nitric acid (from Merck) in high-pressure Teflon vessels and then were diluted with water and neutralised with ammonia. The metal concentrations were then determined as in the culture media. The metal fixed per microalgal cell ([M] cell ) was calculated from the [M] algae and cell counts. Control filters were processed using the same procedure, but the metal contamination was found to be insignificant (< 1%). [M] algae approximately balanced the metal lost from the seawater (with an error < 5 %), confirming that metal adsorption onto the polycarbonate culture bottles was negligible. Reference standard seaweed (CRM 279 Ulva lactuca) was used to check the accuracy of the digestion and analysis procedures. The metal contents found were not significantly different from expected values ('t' test, P > 0.05).

Influence of culture media composition on trace metal uptake by P. tricornutum was examined using principal components analysis (PCA) by means of PRIMER software [START_REF] Clarke | Change in Marine Communities: An Approach to Statistical Analysis and Interpretation[END_REF][START_REF] Clarke | Non-metric multivariate analysis in community-level ecotoxicology[END_REF].

Results and Discussion

Changes observed in the culture media of different algal species

A complete characterization of the media in which micro-and macroalgae were incubated could not be performed due to technical limitations. However, levels of total organic ligands, C L (evaluated as being those capable of strong complexation with Cu(II)), trace metals and cysteineand glutathione-like thiols were determined. Thiols may be very relevant components of these media. In fact, previous work carried out with similar culture media has shown that thiols (that is, compounds identified as being cysteine or glutathione in CSV, because the respective peak coincided with that of the thiol standard added for calibration) accounted with about 87 % of the total dissolved organic ligands (Vasconcelos et al., 2002). In addition, statistically significant correlations between C L and thiols were recorded by Vasconcelos et al. (2002), suggesting that these thiolic compounds may be major contributors to the organic ligands released by eukaryotic algae. There is evidence that sulfur-containing compounds of low molecular mass are important components of the pool of ligands responsible for the nearly complete complexation of Cu in surface seawater. Free cysteine is known to occur in seawater (van den Berg et al., 1988).

Compounds identified as glutathione (γ-glutamylcysteinylglycine) have also been found in seawater (Le Gall and van den Berg, 1993) and estuarine waters [START_REF] Tang | The distribution of biogenic thiols in surface waters of Galveston Bay[END_REF][START_REF] Laglera | Copper complexation by thiol compounds in estuarine waters[END_REF][START_REF] Tang | Factors affecting the presence of dissolved glutathione in estuarine waters[END_REF]. Therefore, despite other compounds that may be released by the algae, the chemicals measured in the present work seems to be pertinent to the interpretation of the results.

tricornutum displayed higher concentrations of the determined exudates than E. Huxleyi did, despite the same numbers of cells in the inoculation. The macro-alga Enteromorpha also produced more exudates than Porphyra, although the initial algal mass was the same. The composition of the identified exudates varied with the alga species, although the overall conditional stability constants of the respective Cu-complexes were similar in all media (log K' Cu(II)L between 12.07 and 12.65).

Glutathione-like compounds were released in all media by the following increasing order: P.

tricornutum < E. huxleyi < Porphyra < Enteromorpha, whereas cysteine-like compounds were released only in media in which micro-organisms were incubated, the concentration being about 7 times higher for P. tricornutum culture. It should be noted that in the media enriched with P. tricornutum exudates the total concentration of Cu-complexing organic ligands, C L , was markedly lower than the sum of the concentrations of cysteine-and glutathione-like compounds. This result may account the fact of the Cu:thiol stoichiometric ratios can be either 1:2 (Leal and van den Berg, 1998) or 1:1 (Le Gall and van den Berg, 1993), depending on the metal to ligand concentration ratio in the medium.

Production and liberation of complexing substances by phytoplankton has frequently been related to the deficiency or excess of trace metal levels, in comparison to levels common in natural seawater. For instance, liberation of glutathione by the diatom T. weissflogi [START_REF] Tang | Factors affecting the presence of dissolved glutathione in estuarine waters[END_REF][START_REF] Tang | Response of nonprotein thiols to copper stress and extracellular release of glutathione in the diatom Thalassiosira weissfligii[END_REF] or cysteine by E. huxleyi [START_REF] Dupont | Novel copper-binding and nitrogen-rich thiols produced and exuded by Emiliania huxleyi[END_REF][START_REF] Dupont | Effects of copper, cadmium, and zinc on the production and exudation of thiols by Emiliania huxleyi[END_REF] was found to increase in response to an increase of Cu in the medium. In response to a rise in Cu, Cd or Zn concentrations, exudation of arginine-cysteine, glutamine-cysteine, cysteine, glutathione or γglutamate-cysteine by the coccolithophorid huxleyi have been recorded [START_REF] Dupont | Effects of copper, cadmium, and zinc on the production and exudation of thiols by Emiliania huxleyi[END_REF].

On the other hand some compounds are released in response to a deficiency in micronutrients, as is the case with phytosiderophores released by plants in response to a deficiency in Fe or Zn [START_REF] Reichard | Goethite Dissolution in the Presence of Phytosiderophores: Rates, Mechanisms, and the Synergistic Effect of Oxalate[END_REF]. In the present work the micro-and macro-algae were incubated in coastal

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natural seawater without addition of metals, therefore we can not infer if the trace metal levels available in the media condition the nature and concentration of exudates.

Incubation of the micro-and macro-algae for 7 days caused some changes in the trace metal contents of the respective media. Levels of Pb and Fe decreased slightly but significantly in all cultures, with greater reduction in Pb in the medium incubated with E. huxleyi, and Fe in the medium incubated with P. tricornutum. For Mn the more relevant change occurred in the medium containing Enteromorpha, which displayed a very marked increase of Mn levels. The levels of Zn decreased significant and markedly for E. huxleyi and P. tricornutum and to a lesser extent for Porphyra, but in contrast, increased considerably in the presence of Enteromorpha which released Zn into the medium. Therefore, Enteromorpha was the only alga that could release metals (Mn and Zn) into the medium and did not remove any metal from the medium. This alga can bioaccumulate metal, being used as metal bioindicator [START_REF] Say | Enteromorpha as a Monitor of Heavy Metals in Estuarine and Coastal Intertidal Waters[END_REF][START_REF] Leal | Biomonitoring with Benthic Macroalgae and Direct Assay of Heavy Metals in Seawater of the Oporto Coast[END_REF]. This is compatible with its ability to liberate metals in seawater probably poorer in metals than that present at the site from which the alga were collected. Levels of Cu, Cd and Ni did not change significantly in any media relative to the initial seawater composition.

Comparison of growth, exudation and trace metal sorption of P. tricornutum in different media

Growth

The cell yield of P. tricornutum attained a maximum after about 7 d incubation in most of the media (see Fig. 1). Similar growth profile curves for P. tricornutum in f/2 medium were observed previously by [START_REF] Kudo | Chemical fractionation of phosphorus and cadmium in the marine diatom Phaeodactylum tricornutum[END_REF].

Table 1 shows that all the media in which P. tricornutum was incubated promoted cell yield.

In addition, all but the medium containing the exudates from P. tricornutum could promote slightly higher growth rate. These increased by the order (day -1 ): natural seawater = with P. tricornutum exudates (0.909) < with E. huxleyi exudates (0.971) < with Porphyra exudates (1.17) < with

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Enteromorpha exudates (1.28). These are only indicative values as the statistical mean of rates is questionable because the calculation was based on only 2-3 points. However, the cell yield followed the same order, and it was identical for E. huxleyi and Porphyra exudates. The P. tricornutum yield in natural seawater was similar to that observed before [START_REF] Kudo | Chemical fractionation of phosphorus and cadmium in the marine diatom Phaeodactylum tricornutum[END_REF] in similar experimental conditions. Promotion of P. tricornutum growth in the media in which different algae had been incubated was as follows: (a) lower or higher levels (depending on the medium) of some metals relative to those present in the natural water (Table 1) did not limit growth or were toxic to P. tricornutum; (b) the presence of exudates of the micro-or macro-algae control metal toxicity and direct or indirectly promoted the P. tricornutum growth.

Exudates of E. huxleyi slightly promoted growth of P. tricornutum (Table 1) whereas in a previous study, in which E. huxleyi has been incubated in seawater enriched in exudates of P. tricornutum and with similar trace metal levels, a drastic inhibition of E. huxleyi growth was observed (Vasconcelos et al., 2002). These contrasting results indicate that the nature and effects of chemicals released by eukaryotic algae can vary markedly among species. P. tricornutum grows well in the presence of E. huxleyi exudates, but E. huxleyi is inhibited by P. tricornutum, therefore the simultaneous presence of both algae in a natural site may lead to the disappearance of E. huxleyi, thus reducing biodiversity.

Trace metal contents

Table 1 also presents the initial total dissolved metal concentrations, [M] d , and the concentrations after 7 d growth in all tested culture media, as well as the percentage of each metal that was transferred to the cells (surface adsorption and uptake). As the cell yields were not identical in the different cultures, the metal fixed per cell, [M] cell , was included in Table 1. Note that the total metal content measured in the cells was statistically identical to the difference between the initial and final [M] d measured in the culture medium.

Table 1 shows that Cu fixed per cell was similar or even higher in the presence of higher concentrations of exudates that are strong Cu ligands, indicating that algal exudates did not reduce the bioavailability of Cu, on the contrary exudates promoted uptake in some cases. The more accentuated effect occurred in the culture with Enteromorpha exudates, where [Cu] cell was 60 % higher than in the control culture. This was in spite of the initial [Cu] d which was 20 % lower (i.e. there was less Cu available per cell) and the final number of cells was 74 % higher, causing much higher competition for the available Cu. These results indicated that the algal exudates directly (e.g. forming lipophilic organic complexes) or indirectly (by unknown mechanisms) promoted the fixation of Cu (and other metals, see below) to P. tricornutum. Concomitantly, in the culture with Enteromorpha exudates there was also uptake of organic ligands (L), particularly glutathione-like compounds. These results are consistent with those previously reported multispecies algal bioassays for assessing Cu toxicity, involving three marine microalgae (Micromonas pusilla, P. tricornutum, and Heterocapsa niei) [START_REF] Franklin | Development of multispecies algal bioassays using flow cytometry[END_REF]. Cu toxicity to P. tricornutum was reduced, compared to single-species bioassays, and such reduction could not be explained by differences in the Cu complexing capacity in solution (as a result of exudates production) because labile Cu, measured by anodic stripping voltammetry, has been similar for P. tricornutum alone and in the mixture.

In addition, in the presence of Enteromorpha exudates the amount of Ni fixed per cell,

[Ni] cell , was double that fixed in the presence of Porphyra exudates (4.0 x 10 -18 vs. 2.0 x 10 -18 mol cell -1 ), even though the initial [Ni] d was similar in both media and the final cells number was 30 % lower in the last case, resulting in more [Ni] d available per cell. Higher cellular metal levels in the presence of Enteromorpha exudates were also found for Cd. Together, these results indicated that Enteromorpha exudates promoted the fixation of Cu, Cd and Ni in P. tricornutum. The simultaneous uptake of glutathione like compounds, 66 % of the initial values, seems to be implicated in higher metal sorption.

Remarkable results were also observed for [Zn] cell and [Mn] cell in the presence of Enteromorpha exudates, where [Zn] cell was only 41 x 10 -18 mol cell -1 , much lower than that in the control culture, 109 x 10 -18 mol cell -1 , despite the initial [Zn] d was more than two times higher in the culture enriched with Enteromorpha exudates (139 nmol L -1 vs. 59 nmol L -1 in the control). The lower [Zn] cell was not caused by scarcity of dissolved Zn since the final [Zn] d was double of the initial [Zn] d in the control medium. The presence of relatively high levels of glutathione-like compounds may prevent Zn sorption. Gnassia-Barelli and Romeo (1987) found that Zn toxicity has been alleviated by the addition of algal exudates (aged cultures). [START_REF] Stauber | Mechanism of toxicity of zinc to the marine diatom Nitzschia closterium[END_REF] found that most of the zinc associated with the cells of the marine diatom Nitzschia closterium were bound at the cell surface with only 3 to 4 % of this extracellular zinc penetrating the cell membrane. In contrast to the Zn results, [Mn] cell was much higher in the presence of Enteromorpha exudates (54 x 10 -18 mol cell -1 ), where the initial [Mn] d was also higher (75 nmol L -1 ) (in the control culture [Mn] cell = 4.1 x 10 -18 mol cell -1 and the initial [Mn] d was only 6.6 nmol L -1 ). Therefore, in the presence of relatively high levels of both Zn and Mn (and Enteromorpha exudates) the cells of P. tricornutum sorbed preferentially Mn. This is compatible with the hypothesis that Mn could replace Zn in some cellular functions. A relationship between Mn and Zn has also been observed for other algae. For example, [START_REF] Sunda | Interactions among Cu 2+ , Zn 2+ , and Mn 2+ in controlling cellular Mn, Zn, and growth rate in the coastal alga Chlamydomonas[END_REF] observed that up to 150 nmol L -1 [Zn] d inhibited cellular Mn uptake by the coastal alga Chlamydomonas sp. Nevertheless, this result contrasts with that observed in the present work. However, the previous work was carried out in absence of algal exudates and the culture medium contained a chelator (NTA or EDTA) as a trace metal buffer.

Therefore, the composition of the culture medium (and the algal species) was very different from those used in the present work.

Other important findings were as follows. The exudates of all tested algae caused inhibition of Fe transfer to the P. tricornutum cells. For instance, in the presence of Porphyra exudates, the [Fe] cell was 77 % lower than that in the control culture, though the initial [Fe] d was only 11 % lower, and the cell numbers only 35 % higher. It must be noted that the final [Fe] d was much higher in the medium with Porphyra exudates than in the control (14.3 vs. 5.4 nmol L -1 ) indicating that the lower [Fe] cell was not caused by scarcity of Fe in the medium. Although Fe is considered the most important trace metal for plant metabolism, because Fe is required for many proteins, enzymes and reactions [START_REF] Kudo | Combined effects of temperature and iron on the growth and physiology of the marine diatom Phaeodactylum tricornutum (Bacillariophyceae)[END_REF], the lowest level of Fe per cell was observed in the culture which displayed the highest yield (and rate of growth). Possibly uptake of other elements could compensate for the decrease of cellular Fe.

To obtain a broader view of the influence of medium composition on P. tricornutum behaviour, PCA analysis based on trace metal uptake was carried out. Calculations were performed either for percentage of metal removed from the initial solution (% uptake in Table 1) or for metal taken up per cell, and results are plotted in Fig. 2a and 2b respectively. For comparison, the influence of similar culture media on E. huxleyi behaviour observed before (Vasconcelos et al., 2002) was also analysed (Fig. 2c and2d). For P. cultures, the first two principal axes retained 80.5 % (Fig. 2a) or 85.8 % (Fig. 2b) of the variance from the initial seven dimensions. In For E. huxleyi cultures, higher differences occurred between P. tricornutum exudates and control grouped with exudates of its own alga (PC1, Fig. 2c, where the first two principal axes retained 84.3 % of the variance from the original seven dimensions); PC2 separates the group formed by Enteromorpha and Porphyra exudates from the other media. Fig. 2d, where the first two principal axes retained 93.6 % of the variance from the original seven dimensions, shows that P. tricornutum exudates caused metal uptake profiles very different from the other media.

Therefore, independent of the units used in the estimation of metal sorption, PCA analysis confirmed that Enteromorpha exudates medium, in addition to promoting growth of P. tricornutum cells more than the remaining media, caused a very specific profile of metal sorption which did not depend only on the total dissolved metal concentration in the media. PCA revealed that profiles of metal sorption in the presence of E. huxleyi exudates were also very different than those for all the other media.

Enteromorpha and Porphyra exudates acted similarly to E. huxleyi causing metal sorption profiles distinct from the other media, but the most marked uptake profile occurred in the presence of P. tricornutum exudates, which could inhibit E. huxleyi growth (Vasconcelos et al., 2002).

Release and/or uptake of organic ligands in the different media

The values of total final concentrations of organic ligands (C L ) released or taken up per cell of P. tricornutum in the different media are shown in Fig. 3a (see total C L in the solutions in Table 1).

The maximum production of organic ligands per cell of P. tricornutum occurred in the control medium. Therefore, exudates of all tested algal species inhibited the release of organic ligands by P. tricornutum, suggesting that exudates are released either to control excessive levels of some chemicals, like trace metals, or to promote uptake of nutrients. In addition, in the presence of Porphyra or Enteromorpha exudates, about 50 % of the organic ligands initially present in the media were taken up by P. tricornutum cells (in the free form or bound to trace metal ions) during the 7 days of growth. These results were in contrast to those previously observed in cultures of E.

Huxleyi (Vasconcelos et al., 2002), apart from the control media (Fig. 3b). In fact, in the media enriched with exudates of different algal species E. huxleyi liberated more exudates than in the natural seawater. In addition, in the presence of the exudates of P. tricornutum the amount of exudates released by E. huxleyi was about five times higher than that liberated into the other media.

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This result has been interpreted as a defence mechanism against toxicity induced, direct or indirectly, by the P. tricornutum exudates, which inhibited the growth of E. huxleyi. Therefore, the presence in the medium of exudates of certain algal species can affect the extent of exudation of other algae species in a very specific way. This may happen even when accentuated changes in the algal growth yield (stimulation or inhibition) are not detected, as was the case for the present P. tricornutum cultures in the present work.

The concentrations of the combined thiol compounds quantified in the different cultures were directly related to that of the total Cu ligand concentration present in the media (Table 1):

[Thiols] t = (1.06 ± 0.07) C L -(12 ± 9), R 2 = 0.84. The similar magnitude and the covariation of the total organic ligand and total thiol concentrations suggests that these thiols may be major contributors to the organic Cu binding ligands released by P. tricornutum and other eukaryotic algae. In the present case, the measured thiols account for about 94 % of the total Cu organic ligands. Similar results were found in a previous study with E. huxleyi (Vasconcelos et al., 2002) and confirms the importance of thiols as complexing agents in the tested cultures, and probably in natural systems where the producer algae are abundant. Fig. 4a shows that cysteine-like compounds are released by P. tricornutum in all tested media (see also Table 1). The highest production during the growth (from 3.5 nmol L -1 to 92 nmol L -1 ) occurred in the natural seawater (control medium) and the lowest in the medium enriched with P. tricornutum exudates (from 92 nmol L -1 to 106 nmol L -1 ). Apparently, the already relatively high initial concentration of cysteine-like compounds inhibited the release of these thiols by P.

tricornutum.

In contrast, the concentration of glutathione-like compounds decreased by ~ 50 % during the growth of P. tricornutum in all media apart from the control (Fig. 4a), where the concentration of glutathione-like compounds was initially 22 nmol L -1 and increased by ~ 50 % after 7 d (Table 1).

In the cultures enriched with Porphyra and Enteromorpha exudates the initial glutathione-like compounds were relatively high (> 100 nmol L -1 ). Therefore, glutathione removal was high in the ACCEPTED MANUSCRIPT media with high initial concentration of glutathione, which varied as follows: natural seawater < with P. tricornutum exudates < with E. huxleyi exudates < with Porphyra exudates < with Enteromorpha exudates. These thiolic compounds were probably taken up by the cells (see below), which is consistent with the decrease of the total organic ligand concentration in the media, as discussed above. The uptake of the thiolic compounds may be directly or indirectly implicated in the improvement of P. tricornutum cells yield observed in the presence of Porphyra and Enteromorpha exudates.

To ensure that the decrease of thiolic compounds in the media was a result of uptake by the organisms and not caused by degradation, in a previous study by Vasconcelos et al. ( 2002) P. tricornutum exudates, 150 nmol L -1 cysteine and 100 nmol L -1 glutathione were added, separately, to sterilised seawater which was conditioned like the algal cultures for 10 days. It was observed that the thiol concentrations were kept constant over the 10 d period, indicating that the decrease in the present study was really due to uptake by the micro-organisms. In addition, synthetic cysteine and glutathione were added to E. huxleyi cultures, causing changes in the growth similar to those induced by the P. tricornutum exudates (Vasconcelos et al., 2002). These results support the interpretation of the data concerning cysteine-and glutathione-like compounds in the present work.

A comparison of P. tricornutum organic ligand concentrations with those observed before for E. huxleyi in similar experimental conditions (Vasconcelos et al., 2002) (Fig. 4b) demonstrates that nature and intensity of cell exudation is markedly dependent on the algal species. Contrasting results are demonstrated by the effects of P. tricornutum exudates on E. huxleyi cell behaviour (growth inhibition) and the effects of E. huxleyi exudates in the P. tricornutum cell behaviour (growth promotion). Release or uptake of ligands may be related to metal uptake although how and to what extent can not be inferred from the available data.. [START_REF] Ahner | Glutathione and other low molecular weight thiols in marine phytoplankton under metal stress[END_REF] exposed five species of marine algae to variable concentrations of Cd and Cu and measured the intracellular concentrations of glutathione, cysteine, γ-glu-cys and phytochelatin. These authors found that glutathione in E. huxleyi seemed to decrease at high Cu concentrations, while in P. tricornutum it showed an increase at Cu 2+ = 1 nmol L -1 . These results are compatible with those obtained in the present study: P. tricornutum took up an appreciable fraction of the glutathione-like compounds initially present in the media, whereas E.

huxleyi released these thiols into all media. Very high rates of glutathione efflux have been invoked

to explain the rapid increase in dissolved ligand concentrations in E. huxleyi cultures [START_REF] Ahner | Glutathione and other low molecular weight thiols in marine phytoplankton under metal stress[END_REF]. In addition, in response to Cd exposure both cysteine and γ-glu-cys (precursors to glutathione) increased in the presence of E. huxleyi, whereas only γ-glu-cys increased in the presence of P. tricornutum. [START_REF] Kawakami | Effects of metal combinations on the production of phytochelatins and glutathione by the marine diatom Phaeodactylum tricornutum[END_REF] have examined the effects of Cu, Cd and Zn on the intracellular production of the polypeptides phytochelatin and glutathione by P. tricornutum.

Single additions of Cu (0.4 µmol L -1 Cu 2+ ) and Cd (0.45 µmol L -1 Cd 2+ ) to the culture medium induced the production of phytochelatins, whereas a single addition of Zn (2.2 µmol L -1 Zn 2+ ) did not stimulate phytochelatin production. Combination of Zn and Cu resulted in a similar phytochelatin production as compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production. Glutathione concentrations were affected only upon exposure to Cd (85 % increase) or the combination of Cd with Zn (65 % decrease), relative to the control experiment. In some of the cited papers, the production of thiols occurred as a defence mechanism against exposure to relatively high trace metal levels, which was not the case in this work. The present results may be classified as allelopathic effects between algal species, and may take place in multispecies algal cultures or in the real environment, particularly during bloom events. Such phenomena can lead to predominance of some algal species and inhibition of other species. Simultaneously, important changes in chemical speciation of the media and metal uptake by phytoplankton may take place in the aquatic medium.

Conclusion

This work demonstrated that exudates of different marine micro-and macro-algae caused allelopathic effects in P. tricornutum cultures. Cell yield of P. tricornutum was increasingly promoted by exudates of E. huxleyi > Porphyra > Enteromorpha. Although exudates can strongly complex Cu (and probably other metals), their presence promoted Cu uptake. Significant changes of Ni, Cd, Fe, Zn and Mn uptake by P. tricornutum were also observed in the presence of exudates of different algal species. In addition, both intensity of production and nature of exudates released by P. tricornutum were markedly influenced by the presence of exudates of other algae, the allelopathic effects being very specific (variable from one species to another).

The fact that P. tricornutum growth was promoted by macro-algae exudates which usually compete with this diatom (a basically benthic, rock pool species) may not to be entirely realistic.

The present results were obtained in the absence of the exudates producers (living macro-algae),

and it is possible that in a real aquatic system where the diatom and macro-algae exist together, other exudates with inhibitor properties will also be released by the macro-algae. Nevertheless, it is important to stress the fact of exudates of Porphyra and Enteromorpha (which can live together)

gave rise to a markedly different response from the diatom, showing specific behaviour.

Although, allelopathy will occur in the aquatic environment, it will probably be to a lesser extent than in cultures, and primarily during bloom events and may have effects on both chemical speciation and bioavailability of chemicals to phytoplanktonic species. Such changes might cause the dominance of some species over other species. Therefore, in future studies involving in vitro cultures more attention should be paid to the role of algal exudates, in order to improve the significance of the results with the aim of using them as models of the real environment, (10 -18 mol cell -1 ) 3.4 ± 0.4 0.030 ± 0.005 0.021 ± 0.006 4.5 ± 0.5 6.0 ± 0.4 2.0 ± 0.2 1.4 ± 0.3 [cells] (10 6 cell L -1 ): 665 ± 33 P. tricornutum 0 d growth: 5.1 ± 0.2 0.20 ± 0.02 0.14 ± 0.03 5.1 ± 0.4 5.6 ± 0.8 4.8 ± 0.2 4.6 ± 0.5 106 ± 3 92 ± 4 34 ± 2 7 d growth: 3.1 ± 0.1 0.099 ± 0.008 0.097 ± 0.005 1.4 ± 0.2 1.6 ± 0.2 3.9 ± 0.3 2.5 ± 0.2 122 ± 5 106 ± 5 18.2 ± 0.9 Uptake e (%) 39 50 31 72 71 19 46 [M] cell (10 -18 mol cell -1 ) 3.7 ± 0.3 0.19 ± 0.02 0.080 ± 0.005 6.8 ± 0.3 7.4 ± 0.5 1.7 ± 0.3 3.9 ± 0.2 [cells] (10 6 cell L -1 ): 540 ± 27 Porphyra 0 d growth: 6.3 ± 0.3 0.29 ± 0.03 0.18 ± 0.01 25 ± 2 19.0 ± 0.5 6.1 ± 0.3 5.1 ± 0.2 116 ± 3 ---110 ± 6 7 d growth: 4.4 ± 0.2 0.11 ± 0.01 0.14 ± 0.02 13.6 ± 0.5 14.3 ± 0.4 1.8 ± 0.3 3.8 ± 0.1 77 ± 2 13.1 ± 0.5 39 ± 2 Uptake e (%) 30 62 22 46 26 70 25 [M] cell (10 -18 mol cell -1 ) 2.9 ± 0.2 0.28 ± 0.01 0.060 ± 0.002 17 ± 2 7.2 ± 0.6 6.6 ± 0.5 2.0 ± 0.4 [cells] (10 6 cell L -1 ): 653 ± 33 Enteromorpha 0 d growth: 5.4 ± 0.2 0.24 ± 0.03 0.16 ± 0.02 139 ± 5 14.4 ± 0.8 75 ± 4 5.9 ± 0.2 175 ± 4 3.3 ± 0.2 154 ± 8 7 d growth: 1.2 ± 0.2 0.13 ± 0.01 0.052 ± 0.005 104 ± 2 10.3 ± 0.3 29 ± 2 2.5 ± 0.2 123 ± 5 23 ± 1 88 ± 4 Uptake e (%) 78 46 68 25 28 61 58 [M] cell (10 -18 mol cell -1 ) 5.0 ± 0.4 0.13 ± 0.02 0.13 ± 0.01 41 ± 3 4.8 ± 0.2 54 ± 3 4.0 ± 0.2 [cells] (10 6 cell L -1 ): 845 ± 42 a-Mean values ± standard deviation (n = 3). b-The initial P. tricornutum cells concentration was about 1.5 x 10 6 cell L -1 . c-Units: equivalents of nmol L -1 Cu. a-Species determined by CSV as cysteine or glutathione. e-Percentage of the initially soluble metal that was bound up with the cells (extra-and intra-cellularly).

Figure captions

  stock unialgal, axenic, E. huxleyi (Lohmann PCC N o 92 and 92d, isolated by Hay and Muller, Plymouth Marine Laboratory) or P. tricornutum (from the same origin) culture. The initial concentration of algae was approximately 1.5 x 10 6 cells L -1 (P. tricornutum) or 0.5 x 10 6 cells L -1 (E. huxleyi). Algal exudates were obtained by growing up the respective algae for 7 d at 18 ºC under 16 h light (and 8 h dark) (exponential phase). Seawater enriched with Porphyra spp. or Enteromorpha spp. exudates was obtained by submerging a fresh sample of each macroalgae (20 g wet weight of algae per litre of sterilised seawater) in a polycarbonate bottle, for 24 h (time enough for the liberation of a significant amount of exudates but insufficient for the degradation of the macroalgae, Vasconcelos and Leal, 2001) at 18 °C under 16 h light (and 8 h dark). The intertidal benthic macroalgae were collected by hand from the surface of the beach rocks at low tide at Matosinhos beach (Northwestern Portugal). The algae were randomly placed in acid-cleaned plastic bags and transported to the laboratory in seawater collected from the beach. Upon arrival at the laboratory, the algae were washed twice with 3.5 % (w/v) sterilised NaCl (filtered through a 0.1 µm pore-size filter and of concentration identical to that of the natural environment to avoid osmotic crash).

  8 (Ni); t d (s) = 60-120. The scanning parameters were: frequency (Hz): 50 (Cu, Zn), 200 (Pb, Cd, Mn), 10 (Fe, Ni); modulation amplitude (mV): 25 (Cu, Pb, Cd, Mn, Zn), 50 (Fe, Ni); step potential (mV): 2.44 (Cu, Zn), 3.66 (Pb, Cd, Fe, Mn, Ni); modulation time (s): 0.01 (Fe, Ni, Co); interval time (s): 0.1 (Fe, Ni, Co); squarewave (Cu, Pb, Cd, Zn and Mn), differential-pulse (Fe and Ni). In all cases a hanging mercury electrode (HMDE) was used. [M] d was also determined, using the same procedures, in a seawater reference material for trace metals (NASS-5) and the experimental values were found to not be statistically different from the certified values ('t' test, P > 0.05).

  d , so as to prevent dissociation of the CuL complexes The scanning parameters were the same used in the [Cu] d determinations (described above).

Fig. 2a the

  Fig. 2a the first principal component (PC1) clearly separated the control and the medium with P.

Fig. 1 .

 1 Fig. 1. Phaeodactylum tricornutum growth in the different culture media. Standard deviations (n=3)

Fig. 2 .

 2 Fig. 2. Principal component analysis plots for seven metals (Cu, Pb, Cd, Zn, Fe, Mn and Ni) and

Fig. 3 .

 3 Fig. 3. Cu-complexing ligand concentrations released (or taken up) by Phaeodactylum tricornutum

Fig. 4 .

 4 Fig. 4. Comparison of thiol compounds released (or taken up) by Phaeodactylum tricornutum after

  Fig.1

Table 1 .

 1 Trace metal ([M] d ), Cu-complexing ligands (C L ) and thiol ([Cysteine] and[Glutathione]) contents a (nmol L -1 ) in natural seawater, media in which micro-and macro-algae have been incubated and in the P. tricornutum cultures b in media enriched with exudates of different algal species. The numbers of cells at the end of the experiments are also given.

	Medium: Seawater	Cu	Pb	Cd	Zn	Fe	Mn	Ni	C L	c	Cysteine d Glutathione d
	Natural (control)	0 d growth: 6.8 ± 0.2	0.36 ± 0.02	0.19 ± 0.01	59 ± 2	20.5 ± 0.4 6.6 ± 0.7 6.7 ± 0.2 48 ± 3	3.5 ± 0.2	22 ± 1
	7 d growth: 5.3 ± 0.3 Uptake e (%) 22 [M] cell (10 -18 mol cell -1 ) 3.1 ± 0.1 [cells] (10 6 cell L -1 ): 485 ± 24	0.20 ± 0.04 44 0.33 ± 0.02	0.12 ± 0.01 37 0.14 ± 0.02	6.3 ± 0.3 5.4 ± 0.4 4.6 ± 0.3 4.0 ± 0.1 106 ± 3 89 74 30 40 109 ± 5 31 ± 2 4.1 ± 0.1 5.6 ± 0.5	92 ± 4	34 ± 2
	Enriched with exudates of:
	E. huxleyi	0 d growth: 5.2 ± 0.3	0.13 ± 0.01	0.10 ± 0.02	7.8 ± 0.4	15 ± 1	5.2 ± 0.4 4.7 ± 0.2 74 ± 2 12.0 ± 0.6	61 ± 3
		7 d growth: 2.9 ± 0.3 Uptake e (%) 44	0.11 ± 0.01 0.086 ± 0.004 4.8 ± 0.2 15 14 38	11 ± 1 27	3.9 ± 0.1 3.8 ± 0.3 103 ± 6 25 19	61 ± 3	27 ± 4
	[M] cell

Acknowledgements

The Post-Doctoral grant of M. Fernanda C. Leal (PRAXIS XXI / BPD / 22095 / 99) was funded by the Portuguese Foundation for Science and Technology (FCT). Mr. Manuel Isidro from the Aquaculture of Rio Alto, Póvoa do Varzim, is acknowledged for providing facilities for collection of seawater.

ACCEPTED MANUSCRIPT

van den Berg, C.M.G., and Huang, Z.Q., 1984


Enrique Mateos-Naranjo

Susana Redondo-Gómez

Jesús Cambrollé

M Enrique Figueroa

Growth and photosynthetic responses to copper stress of an invasive cordgrass, Spartina densiflora

Keywords: Copper, Growth rate, Invader, Photochemistry, Photosynthesis, Photosynthetic pigments, Salt marshes, Spartina densiflora, Tissue analysis

Spartina densiflora Brongn., is found in coastal marshes of south-west Spain, growing in sediments with between 300 -3000 mg Cu kg -1 total soil DW (450-4500 mg Cu kg -1 supposing that the soil porosity is 0.5). An experiment was designed to investigate the effect of copper from 0 to 5000 mg kg -1 (64 mmol l -1 ) on the photosynthetic apparatus and the growth of S. densiflora. We also determined total ash, copper, calcium, magnesium and phosphorous concentrations, as well as C/N ratio. S. densiflora survived to concentrations as high as 320 mg Cu kg -1 DW in leaves, although excess of Cu diminished water use efficiency and Ca-, Mg-and P-uptake. Also, quantum efficiency of PSII, net photosynthetic rate, stomatal conductance and pigment concentrations declined with increasing external Cu. Finally, the decline in the photosynthetic function resulted in a biomass reduction of between 50 and 80% (for 600 and 5000 mg Cu kg -1 , respectively).

Introduction

Copper is an essential micronutrient for plant growth, participating in important biological reactions as an enzymatic cofactor and electron carrier in the photosynthetic and respiratory processes [START_REF] Andrade | Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro[END_REF] but, when in excess, it becomes highly toxic [START_REF] Dewez | Determination of photosynthetic and enzimatic biomarkers sensitivity used to evaluate toxic effects of copper and fludioxonil in alga Scenedesmus obliquus[END_REF]. The excess of copper reduces plant growth as well as photosynthetic and respiratory activities [START_REF] Nalewajko | Differential responses of growth, photosynthesis, respiration, and phosphate uptake to copper in copper-tolerant and copper-intolereant strains of Scenedesmus acutus (Chlorophyceae)[END_REF]. The photosynthetic apparatus is particularly susceptible to this cation, resulting in a decrease in the activity of photosystem II and electron transfer rates (Fernades and Henriques, 1991;[START_REF] Mallick | Use of chlorophyll fluorescence in metal-stress research: a case study with the green microalga Scenedesmus[END_REF]. Toxic concentrations of Cu may eventually build up in certain estuarine and shallow coastal marine habitats near mining and industrial hot spots [START_REF] Nielsen | Photosynthetic responses to Cu2+ exposure are independent of light acclimation and uncoupled from growth inhibition in Fucus serratus (Phaeophyceae)[END_REF].

The austral cordgrass, Spartina densiflora Brongn. (Poaceae), is invading salt marshes as far apart as southern Europe [START_REF] Tutin | Spartina Schreber[END_REF][START_REF] Mateos-Naranjo | Effect of prolonged flooding on the invader Spartina densiflora Brong[END_REF], North Africa [START_REF] Fennane | Nouveaux materiaux pour la flore de Maroc. Fascicule 3[END_REF] and North America [START_REF] Kittelson | Mechanisms of expansion for an introduced species of cordgrass, Spartina densiflora, in Humboldt Bay, California[END_REF]. This species has proven to be a vigorous invader and ecosystem engineer that spreads by clonal growth and prolific seed production (Mateos-Naranjo et al., 2008a). In the salt marshes of the joint estuary of the Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world [START_REF] Ruiz | Trace metals in estuarine sediments of south-western Spain[END_REF][START_REF] Sáinz | Influence of the very polluted inputs of the Tinto-Odiel system on the adjacent littoral sediments of southwestern Spain: A statistical approach[END_REF] on account of the intense mining activity and waste from industrial zones, S. densiflora grows over sediments with between 300 -3000 ppm Cu per total soil DW [START_REF] Nelson | Heavy metal anomalies in the Tinto and Odiel River and estuary system, Spain[END_REF][START_REF] Sáinz | Characterisation of sequential leachate discharge of mining waste rock dumps in the Tinto and Odiel rivers[END_REF], and with the greatest mobility and bioavailability [START_REF] Morillo | Heavy metal distribution in marine sediments from the southwest coast of Spain[END_REF]. Other species of the Spartina genus have been shown to have a great capacity to accumulate metals, such as copper, from the sediments. The physiological impact of such bioaccumulation, nonetheless, has not been thoroughly studied [START_REF] Nybakken | Marine Biology: an ecological approach[END_REF].

The main objective of the present study was to evaluate the effects of elevated copper levels on S. densiflora growth and photosynthesis. The specific objectives were to: (1) analyze the growth of plants in experimental copper treatments ranging from 0 to 64 mmol l -1 Cu; (2) ascertain the extent to which the effects on the photosynthetic apparatus (PSII chemistry), on gas exchange parameters and on photosynthetic pigments determine plant performance; and (3) examine the possible role of concentrations of mineral matter (ash), calcium, magnesium, phosphorous and nitrogen in explaining the copper effects on growth. ). The pots were carefully irrigated with 20% Hoagland's solution [START_REF] Hoagland | The water culture method for growing plants without soil[END_REF] as necessary. All the pots received the same irrigation.

Material and methods

1. Plant material

Stress treatments

In April 2007, after a month of seedling cultures, the pots were allocated to five Cu The control, 0 mmol l -1 Cu treatment, had exactly 0.0005 mmol l -1 of Cu, since Hoagland's solution contains a small amount of Cu as an essential trace nutrient. Cu concentrations were chosen to cover variations recorded by [START_REF] Nelson | Heavy metal anomalies in the Tinto and Odiel River and estuary system, Spain[END_REF] 300-3000 mg Cu kg -1 per total soil DW, i.e. 450-4500 mg Cu kg -1 supposing that the soil porosity is 0.5) and [START_REF] Sáinz | Characterisation of sequential leachate discharge of mining waste rock dumps in the Tinto and Odiel rivers[END_REF]

Growth

At the beginning and at the end of the experiment (i.e. after 30 d of treatment), four and six entire plants (roots and leaves), from each treatment, respectively, were dried at 80ºC for 48 h and then weighed. Dried, ground samples were ignited in lidded, ceramic crucibles and ash weights were recorded; the furnace temperature was raised slowly over 6 h to 550ºC and this temperature was maintained for a further 8 h. Also, both before and after the copper treatment, the number and height of all fully developed tillers were measured.

The relative growth rate (RGR) in ash-free dry mass of whole plants was calculated by using the formula: RGR = (ln Bf -ln Bi) • D -1 (g g -1 day -1 )

where Bf = final dry mass (an average of the four plants from each treatment dried at the beginning of the experiment), Bi = initial dry mass and D = duration of experiment (days).

Leaf elongation rate (LER) was measured in random leaves (n = 12, per treatment; two measurements per plant) by placing a marker of inert sealant at the base of the youngest accessible leaf. The distance between the marker and the leaf base was measured after 24 h [START_REF] Ewing | A comparison of indicators of sublethal salinity stress in the salt marsh grass, Spartina Patens (Ait.) Muhl[END_REF].

Determination of copper, calcium, magnesium, phosphorous and nitrogen

In accordance with protocols of [START_REF] Redondo-Gómez | Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides[END_REF], at the end of the experiment, leaf and root samples were dried at 80ºC for 48 h and ground. Leaves and roots were carefully washed with distilled water before any further analysis. Then 0.5 g samples (in triplicate), taken from a mixture of the leaves or the roots belonging to the six plants used for each treatment, digested with 6 ml HNO 3 , 0.5 ml HF and 1 ml H 2 O 2 .

Ca, Mg, P and Cu were measured by inductively coupled plasma (ICP) spectroscopy (ARL-Fison 3410, USA). Total N and C concentrations were determined for undigested dry samples by means of an elemental analyzer (Leco CHNS-932, Spain).

Chlorophyll fluorescence

Chlorophyll fluorescence was measured in random fully developed penultimate leaves (n = 12, two measurements per plant) using a portable modulated fluorimeter (FMS-2, Hansatech Instrument Ltd., England) after 7 and 30 d of treatment. Light-and darkadapted fluorescence parameters were measured at dawn (stable, 50 µmol m -2 s -1 ambient light) and at midday (1600 µmol m -2 s -1 ).

The plants were dark-adapted for 30 minutes, using leaf-clips exclusively designed for this purpose. The minimal fluorescence level in the dark-adapted state (F 0 ) was measured using a modulated pulse (<0.05 µmol m -2 s -1 for 1.8 µs) which was too small to induce significant physiological changes in the plant. The data stored were an average taken over a 1.6 second period. Maximal fluorescence in this state (F m ) was measured after applying a saturating actinic light pulse of 15000 µmol m -2 s -1 for 0.7s.

The values of the variable fluorescence (F v = F m -F 0 ) and maximum quantum efficiency of PSII photochemistry (F v /F m ) were calculated from F 0 and F m . This ratio of variable to maximal fluorescence correlates with the number of functional PSII reaction centres, and dark adapted values of F v /F m can be used to quantify photoinhibition [START_REF] Krivosheeva | Cold acclimated and photoinhibition in Scots pine[END_REF].

The same leaf section of each plant was used to measure light-adapted parameters.

Steady state fluorescence yield (F s ) was recorded after adapting plants to ambient light conditions for 30 minutes. A saturating actinic light pulse of 15000 µmol m -2 s -1 for 0.7 s was then used to produce the maximum fluorescence yield (F m ') by temporarily inhibiting PSII photochemistry.

Using fluorescence parameters determined in both light-and dark-adapted states, the following were calculated: quantum efficiency of PSII ( PSII = (F m ' -F s )/ F m '), which measures the proportion of the light absorbed by chlorophyll associated with PSII that is used in photochemistry [START_REF] Maxwell | Chorophyll fluorescence-a practical guide[END_REF]; and non-photochemical quenching (NPQ = (F m -F m ') / F m '; [START_REF] Redondo-Gómez | Growth and photosynthetic responses to salinity in an extreme halophyte, Sarcocornia fruticosa[END_REF], which is linearly related to heat dissipation [START_REF] Maxwell | Chorophyll fluorescence-a practical guide[END_REF].

Gas exchange

Gas exchange measurements were taken on random fully expanded leaves (n = 10, a measurement per plant and four extra measurements taken randomly) 7 and 30 days after the treatment, by using an infrared gas analyzer in an open system (LI-6400, LI-COR Inc., Neb., USA). Net photosynthetic rate (A), intercellular CO 2 concentration (C i ) and stomatal conductance to CO 2 (G s ) were determined at ambient CO 2 concentration of 360 µmol mol -1 , temperature of 20/25ºC, 50 ± 5% relative humidity and a photon flux density of 1000 µmol m -2 s -1

. A, C i and G s were calculated using standard formulae of Von [START_REF] Caemmerer | Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves[END_REF]. The water use efficiency (WUE) was calculated as the ratio between A and transpiration rate [mmol (CO 2 assimilated) mol -1 (H 2 O transpired)].

Photosynthetic pigments

At the end of the experiment period, photosynthetic pigments in fully expanded penultimate leaves (n = 10, a measurement per plant and four extra measurements taken randomly) from each treatment were extracted using 0.05 g of fresh material in 10 ml of 80% aqueous acetone. After filtering, 1 ml of the suspension was diluted with a further

Results

Determination of copper, calcium, magnesium, phosphorous and C/N ratio

There was a marked decrease in the mineral (ash) content of leaves (but not of the roots)

with increasing external Cu concentration (r = -0.76, p < 0.01). The ash content of roots dropped when exposed to 15 mmol l -1 Cu (Fig. 1A). -E).

Carbon/nitrogen (C/N) ratio was higher in the leaves than in the roots of S.

densiflora (Fig. 1F). Both root and leaf C/N ratios tended to increase with external Cu concentration, although leaf ratio dropped sharply when exposed to the highest Cu concentration.

Chlorophyll fluorescence

The fluorescence parameters presented in Figure 2 Furthermore, Fv/Fm values were significantly higher for the control at both dawn and Cu, and the reductions resulted again mainly from the fact that values of Fm were lower at midday than at dawn (t-test, p < 0.05; Fig. 2A). On the other hand, the minimal fluorescence (F 0 ) increased with increasing Cu concentration at both dawn and midday (r = 0.80, p < 0.001; r = 0.82, p < 0.0001, respectively; Fig. 2B), while Fm remained steady. F 0 was inversely correlated with Fv/Fm at both dawn and midday (r = -0.95, p < 0.05; r = -0.93, p < 0.05, respectively).

Quantum efficiency of PSII ( PSII) decreased with increasing Cu concentration, either at dawn or midday (r = -0.88, p < 0.0001; r = -0.75, p < 0.0001; Fig. 2C). PSII values were significantly higher at midday than at dawn.

Non-photochemical quenching (NPQ) did not vary with Cu concentration at dawn, whereas at midday it increased up to 47 mmol l -1 Cu, and then it declined with the further increases in Cu concentration (Fig. 2D).

Gas exchange and pigment concentration

The gas exchange parameters presented in Figure 3 correspond to plants after 30 d of treatment. Net photosynthetic rate (A) after 7 d treatment did not show any differences with the control (c. 15 µmol m -2 s -1 ; p > 0.05) and hence it is not presented here.

Figure 3A shows that A declined sharply with the copper concentration of 15 mmol l -1 and then stabilized at the highest concentrations. There was a strong linear relationship between A and RGR (r = 0.91, p < 0.05), and PSII (r = 0.89, p < 0.05).

Stomatal conductance (Gs) showed the same pattern as A (Fig. 3B). In contrast, intercellular CO 2 concentration (Ci) increased significantly with Cu (Fig. 3C). Water Fig. 2 Fig. 3 use efficiency (WUE), on the other hand, decreased significantly with Cu concentration (Fig. 3D).

Pigment concentrations decreased with Cu concentration, showing the same pattern as A and Gs (Fig. 4).

Growth

Relative growth rate (RGR) was inhibited by Cu excess, as proves the fact that the maximum value was recorded at the control (ANOVA, p < 0.001; Fig. 5A). In addition, Cu treated plants showed chlorosis, especially at the two highest Cu concentrations.

RGR decreased with increasing Cu concentration (r = -0.68, p < 0.0001), and was directly correlated with leaf elongation rate (LER; r = 0.89, p < 0.05), number of tillers (r = 0.95, p < 0.05) and mean height of tillers (r = 0.99, p < 0.001; Figs. 5A-D).

Discussion and conclusions

Spartina densiflora demonstrated a high tolerance to copper stress, since all plants survived even at concentrations as high as 64 mmol l -1 of Cu during 30 d. [START_REF] Paschke | Copper toxicity thresholds for important restoration grass species of the western United States[END_REF] determined the lethal concentrations of copper (LC50, substrate copper concentration that kills 50% of plants) for six grass species used in restoration activities in concentrations close to 5 mmol l -1 after 60 d of treatment. In our glasshouse experiment, we found that Cu levels were much higher in S. densiflora below-(72-1480 ppm Cu DW) than above-ground (10-320 ppm Cu DW) parts. A similar trend was recorded for Spartina maritima [START_REF] Reboredo | Heavy metal analysis of sediments and live tissues of Spartina maritima, from salt marshes of Sado estuary Portugal[END_REF] and Spartina anglica [START_REF] Otte | A survey of zinc, copper, and cadmium concentrations in salt marsh plants along the Dutch coast[END_REF] under field conditions. [START_REF] Luque | Metals in halophytes of a contaminated estuary (Odiel Saltmarshes, SW Spain)[END_REF] found Cu concentrations c. 24 mg kg -1 DW in young leaves of S. densiflora sampled from Odiel salt marshes. In this site, 30 and 200 mg Cu kg -1 DW have been quantified in young and old leaves of this species, respectively; and 150 and 600 mg Cu kg -1 DW for young and old leaves, respectively, from the Tinto Marshes (Huelva, SW Spain; unpublished data). The lower Cu concentration in leaves of S. densiflora, compared to that present in roots, could be accounted for by the development of such mechanisms as compartmentation, which could control the ion transport into leaves, thereby improving its tolerance to heavy metals. The sequestering of metals in tissues or cellular compartments, which are less sensitive to metals, has been described as a tolerance mechanism [START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF], entailing a restriction of the upward movement into the shoots (an avoidance mechanism) and the translocation of excessive metals into the leaves [START_REF] Verkleij | Mechanism of metal tolerance in higher plants[END_REF]). Nevertheless, with excessive levels of soil Cu, this element may be translocated from the roots and accumulated in the tops of the plant (Kabata-Pendias and Pendias, 2001), which could explain the increase of Cu concentration recorded in leaves of S. densiflora with increasing external copper.

On the other hand, roots of S. densiflora could also act as a 'barrier' to the uptake or transport of Cu, since leaf concentration hardly increased when the external concentration exceeded 9 mmol l -1 Cu. In this regard, [START_REF] Verkleij | Mechanism of metal tolerance in higher plants[END_REF] showed that metal-chelating substances may be present in plant exudates, which act to decrease metal uptake, and consequently toxicity. In other species of Spartina, some authors have observed the formation of an iron plaque on the roots, with the ensuant accumulation of metals in the rhizosphere [START_REF] Doyle | Organism-induced accumulation of iron, zinc and arsenic in wetlands soils[END_REF], this plaque being present as well in S.

densiflora [START_REF] Mateos-Naranjo | Growth and photosynthetic responses to zinc stress of an invasive cordgrass, Spartina densiflora[END_REF]. On the other hand, the metal excess in the root zone may inhibit water uptake, thus explaining the recorded decline of water use efficiency.

Due to the significant functions of Cu in enzymes and its variable valence, ions with an affinity to proteins and other compounds similar to that of Cu may have antagonistic interrelationships, a fact which could be the explanation for the decrease in ash content (Kabata-Pendias and Pendias 2001). However, a rise in ash concentration was recorded in roots of S. densiflora at the highest Cu concentration, which could be explained by the strong accumulation of this metal, as the results of internal metal concentrations confirmed. In our experiment, the presence of Cu affected the concentration of the macroelements, Ca, Mg, N and P. An excess of Cu inhibits the activity of phosphatase, thereby diminishing the availability of P [START_REF] Tyler | Heavy metal pollution, phosphatase activity and mineralization of organic phosphorous in forest soil[END_REF]. [START_REF] Lin | Effect of copper concentration on mineral nutrient uptake and copper accumulation in protein of copper-tolerant and non-tolerant Lotus purshianus L[END_REF] found that in Lotus purshianus, an excess of Cu reduced the concentration of P in both root and leaf tissues. Cu-Ca interactions are highly complex and apparently cross-linked with the range of pH in the growth media [START_REF] Kabata-Pendias | Trace elements in soils and plants[END_REF], and Mg is replaced in the chlorophyll by Cu [START_REF] Küpper | Heavy metal-induced inhibition of photosynthesis: targets of in vivo heavy metal chlorophyll formation[END_REF]. [START_REF] Llorens | Effects of copper exposure upon nitrogen metabolism in tissues cultured Vitis vinifera[END_REF] also reported an enhancement of Cu, in respect to N, due to changes in nitrogen metabolism with a reduction of total nitrogen in plant tissues. However, a rise in N concentration was recorded in leaves of S. densiflora at the highest Cu concentration. In this regard, [START_REF] Kabata-Pendias | Trace elements in soils and plants[END_REF] explained that the concentrations of the two elements are relative to the formation of strong protein complexes with Cu.

Furthermore, it is possible that S. densiflora leaves became less rigid at high Cu levels, since [START_REF] Fernandes | Biochemical, physiological and structural effects of excess copper in plants[END_REF] reported that the high sensitivity of diatoms to Cu results from competition between Cu and Si(OH) 4 for the same transport site.

Excessive Cu accumulated in plants tissue can be toxic to plants, affecting several physiological and biochemical processes [START_REF] Balsberg Pahlsson | Toxicity of heavy metal (Zn, Cn, Cd, Pb) to vascular plants[END_REF]. [START_REF] Ouzounidou | Copper-induced changes on growth, metal content and photosynthetic function of Alyssum montanum L. plants[END_REF] explained that the marked impact of copper could be attributed to an overall inhibition of photosystem II (PSII), due to chloroplast membrane leakage; this may be associated with a substantial stress response. In this manner, F v /F m decreases are more permanent than increases of dynamic inhibition ' PSII'; the latter usually relaxes as light intensity is lowered. F v /F m did show a significant reduction at midday compared to dawn values. At midday, the reduction in F v /F m values indicated that S. densiflora experienced photodamage at the highest light flux. PSII decreased as a consequence of the decrease in photochemical quenching (qP) and the increase in NPQ (up to 47 mmol l -1 ), which indicates that the plants dissipated light as heat, in this way protecting the leaf from light-induced damage [START_REF] Maxwell | Chorophyll fluorescence-a practical guide[END_REF]. Nonetheless, at the highest Cu concentration, NPQ diminished, indicating that excess levels of Cu affected this mechanism associated with the adaptation to high light irradiances [START_REF] Demmig-Adams | Survey of thermal energy dissipation and pigment composition in sun and shade leaves[END_REF].

The decline of F v /F m in the presence of copper resulted from an increase in F 0 , while Fm was maintained at a stable level. F 0 increased despite the decline of chlorophyll concentrations, these results being similar to results obtained by [START_REF] Ralph | Photosynthesis response of Halophila ovalis to heavy metals stress[END_REF]. The higher values of F 0 indicated an impact on the PSII reaction centre, as well as to reduction in the transfer of energy from the collecting antennas to reaction centres [START_REF] Ralph | Photosynthesis response of Halophila ovalis to heavy metals stress[END_REF]. [START_REF] Padinha | Evaluating environmental contamination in Ria Formosa (Portugal) using stress indexes of Spartina maritima[END_REF] also found that photosynthetic efficiency was lower in plants of Spartina maritima from polluted sites.

On the other hand, Cu had a negative effect on net photosynthetic rate (A) and stomatal conductance (Gs), without direct relation between both parameters since there was not a reduction in intercellular CO 2 concentration (Ci). [START_REF] Burzynski | Effects of copper and cadmium on photosynthesis in cucumber cotyledons[END_REF] found that lower Gs in cotyledons of Cucumis sativus treated with copper did not influence A, as Ci remained unchanged, and was high. The decrease of A by Cu was attributed to the influence of this metal on the activities of photosynthetic carbon reduction cycle enzymes (PGK and GAPDH were inhibited by Cu), as well as loss of chlorophyll pigments [START_REF] Ouzounidou | Copper-induced changes on growth, metal content and photosynthetic function of Alyssum montanum L. plants[END_REF]. [START_REF] Burzynski | Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress[END_REF] suggested that Cu initially destroys the photosynthetic carbon reduction cycle and subsequently influences the photosynthetic electron transport. In the case of S. densiflora, the decrease in pigment concentrations or increase in its degradation, and consequent negative effect on photosynthetic electron transport, could lead as well to a decline in the photosynthetic function. The substitution the central magnesium ion in the chlorophyll by Cu may also damage the chlorophyll synthesizing system [START_REF] Küpper | Heavy metal-induced inhibition of photosynthesis: targets of in vivo heavy metal chlorophyll formation[END_REF]. In this respect, we recorded a decrease of Mg, whereas copper content increased in the leaf tissue.

The decline in the photosynthetic function resulted in a biomass reduction of S.

densiflora which was apparent in the RGR of ash-free dry mass, number of tillers, leaf elongation rate and mean height of tillers. Thus, the growth of S. densiflora was inhibited by leaf tissue concentrations above 0.03 mg g -1 dry mass.

Spartina densiflora survived to concentrations up to 320 mg Cu kg -1 DW in leaves, when it was treated with 5000 mg Cu l -1 (64 mmol l -1 ), despite the fact that concentrations of Cu between 20 and 100 mg Cu kg -1 DW in mature leaf tissue are considered excessive or toxic [START_REF] Kabata-Pendias | Trace elements in soils and plants[END_REF]. Copper inhibited water use efficiency, Ca-, Mg-and P-uptake, quantum efficiency of PSII, net photosynthesis, stomatal conductance, and pigment synthesis, this resulting in a decline of growth. The analysis was carried out on an ash-free dry mass basis.

  Seeds of S. densiflora were collected in December 2006 from the Odiel Marshes (Huelva, SW Spain), and subsequently stored at 4ºC (in darkness) for three months. After the storing period, seeds were placed in a germinator (ASL Aparatos Científicos M-92004, Madrid, Spain) and subjected to an alternating diurnal regime of 16 hours of light (photon flux rate, 400-700 nm, 35 mol m -2 s -1 ) at 25ºC and 8 hours of darkness at 12ºC, for a month. The seedlings were planted in individual plastic pots (11 cm of diameter) filled with pearlite and placed in a glasshouse (during spring 2007) with minimum-maximum temperatures of 21-25ºC, 40-60% relative humidity and natural daylight (minimum and maximum light flux: 200 and 1000 mol m -2 s -1

  treatments in shallow trays (ten pots per tray, with one tray per Cu treatment): 0, 9, 15, 47 and 64 mmol l -1 Cu, in the same glasshouse. Cu treatments were established by combining 20% Hoagland's solution and CuSO 4 •7H 2 O of the appropriate concentration.

  in the salt marshes of the joint estuary of the Tinto and Odiel rivers. At the beginning of the experiment, 3 L of the appropriate solution were placed in each of the trays down to a depth of 1 cm. During the experiment, the levels in the trays were monitored and they were topped up to the marked level with 20% Hoagland's solution (without additional CuSO 4 •7H 2 O) as a way to limit the change in the Cu concentration due to water evaporation of the nutritive solution. In addition, the entire solution (including CuSO 4 •7H 2 O) was changed on a weekly basis (The total duration of the experiment was 30 d) The possibility of adding NaCl to the culture medium was disregarded, since the absence of salt has been proven to affect neither the photosynthetic function of S. densiflora nor its growth. In fact, Mateos-Naranjo et al. (2008b) found the highest rates of net photosynthesis and leaf elongation in distilled water.

2

  photosynthetic rate data) in order to normalize the error distribution for ANOVA.

  Tissue copper concentrations were greater in the roots than in leaves (two-way ANOVA, p < 0.001), and increased with external Cu concentration (r = 0.89, p < 0.0001; r = 0.98, p < 0.0001, for root and leaf, respectively; Fig 1B). In contrast, leaf and root Ca, Mg and P concentrations diminished with increasing Cu concentration (Figs. 1C

  correspond to plants after 30 d of treatment. Fluorescence parameters after 7 d treatment did not show any difference with the control and are not presented.Values of Fv/Fm diminished, either at dawn or midday, with increasing Cu concentration (dawn: r = -0.85, p < 0.001; midday: r = -0.68, p < 0.001; Fig.3A).

Fig. 1 midday

 1 Fig. 1

  Fig.4

Fig 1 .

 1 Fig 1. Ash (A), total copper (B), total calcium (C), total magnesium (D) and total

Fig 4 .

 4 Fig 4. Chlorophyll a, chl a (A), Chlorophyll b, chl b (B), and carotenoid, Cx+c (C)

  Fig.1 2

Acknowledgements

We are grateful to Mr. F. Fernández-Muñoz for technical assistance. We also thank the Spanish Science and Technology and Environmental Ministries for their support (projects 04212007 and PCI2006-A7-0641, respectively), and Seville University Glasshouse General Service for collaboration.