Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots

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1 Plant Physiology and Biochemistry 41 (2003) Original article Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots Yi-Chieh Chang a, Mohamed Zouari a, Yolanda Gogorcena a, Juan José Lucena b, Javier Abadía a,* a Department of Plant Nutrition, Estación Experimental de Aula Dei, C.S.I.C., Apdo. 202, Zaragoza, Spain b Departamento de Química Agrícola, Geología y Geoquímica, Universidad Autónoma de Madrid, Madrid, Spain Received 18 March 2003; accepted 25 July 2003 Abstract The effects of the heavy metals Cd and Pb on the activity of the enzyme ferric chelate reductase (FC-R, E.C ) have been studied in excised sugar beet root tips. The activity of this enzyme is markedly increased by iron deficiency. Metals were used as chloride salts or chelated with EDTA, and chemical speciation was carried out to predict the metal chemical species in equilibrium both in the ferric reductase assay and in the nutrient solutions. Three different heavy metal treatments were used. First, effects of Cd and Pb on the functioning of the FC-R were assessed in Fe-deficient plants, by including metals in the enzyme assay medium only. Results indicate that 50 µm CdCl 2 or Cd-EDTA did not affect FC-R activities even when assay time was as long as 2 h, whereas Pb slightly decreased enzyme activity only at concentrations of 2 mm. Second, short-time Cd and Pb pre-treatments (30 60 min) were imposed on intact Fe-deficient plants before carrying out the assay of FC-R activity. These short-term treatments induced significant decreases in the FC-R activities previously induced by Fe deficiency. With Cd, effects were more pronounced at higher concentrations, and they were stronger when Cd was in the free ion form than when present in the form of Cd-EDTA chelate. Third, prolonged Cd and Pb treatments were imposed on plants grown on 45 µm Fe-EDTA to assess the long-term effects of heavy metals on the induction of the FC-R enzyme. These long-term heavy metal treatments caused a significant increase in the root FC-R activities, indicating that Cd and Pb induce a deficiency in Fe in sugar beet that in turn elicits FC-R activity. The increases, however, are not as large as those found in total absence of Fe Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cadmium toxicity; Ferric chelate reductase; Heavy metals; Iron chlorosis; Iron deficiency; Lead toxicity; Sugar beet 1. Introduction Heavy metals, coming from natural soil components or from external pollutant sources, may cause serious problems in crops when present in chemical forms available to plants. Decreases in plant growth [9,10], photosynthetic efficiency [6,11,16], nitrate reductase activity [8,17,18], and leaf chlorophyll concentration [4,5] have been reported. Decreases in leaf chlorophyll concentration caused by heavy metals are generally attributed to an induced Fe deficiency [24]. Processes underlying heavy metal-induced Fe Abbreviations: BPDS, bathophenanthroline disulphonate; FC-R, ferric chelate reductase enzyme; PAR, photosynthetic active radiation; PPFD, photosynthetic photon flux density. * Corresponding author. address: jabadia@eead.csic.es (J. Abadía). deficiency have not been fully elucidated yet, but it has been suggested that heavy metals may interfere with root Fe uptake and long distance transport [4,5]. Iron exists in the soil mostly as ferric hydroxide forms, whereas root Fe uptake occurs through divalent metal transporters in the plasma membrane [7,21,23]. Dicotyledonous and non-grass monocotyledonous plants use a plasma membrane enzyme, ferric chelate reductase (FC-R), to reduce Fe(III) to Fe(II). Iron deficiency triggers the induction of the FC-R activity at the root plasma membrane, the development of transfer cells, and the enhancement of the extrusion by roots of protons and different compounds, including reducing substances [14]. Protons secreted by the H + ATPase also help to create an environment beneficial for the FC-R enzyme, which is more active at acidic than at basic ph values [19]. Previous studies indicated that the induction and function of FC-R can be negatively influenced by heavy metal treat Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi: /j.plaphy

2 1000 Y.-C. Chang et al. / Plant Physiology and Biochemistry 41 (2003) ments [1]. Nevertheless, how heavy metals act on the enzyme is yet to be understood. Transporters are also likely to be affected by heavy metals, since the IRT1 transporter responsible for Fe(II) uptake also imports Zn, Mn, Co and Cd [21], whereas the NRAMP high-affinity transporter also imports Cd [23]. In this study, we describe the effects of Cd and Pb, two common heavy metals released in the environment, on the activity of the enzyme FC-R, which is induced under Fe deficiency in sugar beet. Experiments have been carried out with three types of heavy metal treatments. First, the FC-R activity of excised root tips from Fe-deficient plants was measured in the presence of the heavy metals Cd and Pb in the assay solution, in order to determine their effect on the FC-R during the assay. Second, short-time Cd and Pb pretreatments were performed before carrying out the assay of FC-R activity to assess the effect of heavy metals on the activity of the FC-R enzyme induced by Fe deficiency. Third, prolonged Cd and Pb treatments were performed to assess the long-term effects of heavy metals on the induction of the FC-R enzyme in the presence of Fe in the nutrient solution. 2. Results Sugar beet root FC-R activities were assayed with the excised root tip technique first proposed by Moog et al. [15]. The root FC-R activity values obtained were higher with Fe-deficient roots ( nmol Fe g 1 FW min 1 ) than with controls (10 20 nmol Fe g 1 FW min 1 ) (NB1 NB4 genotype) (Fig. 1). This confirms previous results obtained with intact sugar beet plants [19]. however, there was a significant FC-R activity at high ph (Fig. 1), conversely to what occurs in whole roots of intact plants [19]. The FC-R values of Fe-deficient roots often depend on the assay time used, because absorbance may not increase linearly with time (Fig. 2). This was particularly evident at low ph values, which led to large FC-R activities (Fig. 2A). Readings at 5 min in Fig. 2A are equivalent to 225 and 108 nmol Fe g 1 FW min 1 for ph 5.2 and 7.2, respectively. In the sugar beet hybrid Monohil no significant differences in FC-R values were found between reactions carried out at ph 5.5 and 6.0 with (Fig. 2B) or without Hoagland solution (Fig. 2C). Values of root FC-R measured at 5 and 15 min in Fig. 2B were approximately 165 and nmol Fe g 1 FW min 1, respectively. Therefore, in most experiments an assay time lower than 10 min was used to obtain maximal FC-R values. Root tips excreted Fe-reducing compounds during the assay. To ascertain the significance of this excretion, root tips from Fe-deficient plants were incubated in assay medium without bathophenanthroline disulphonate (BPDS) and Fe(III) EDTA, and the supernatant was supplemented with Fe(III) EDTA and BPDS after removal of root tips to assess the extent of the reducing capacity. This reduction was equivalent to approximately 6.7 and 10.4 nmol Fe g 1 FW min 1 at ph values 5.5 and 7.0, respectively (not shown). Therefore, reduction by excreted compounds could account in the Fe-deficient roots for approximately 5 10% of the total FC-R values Characteristics of excised root FC-R: ph and assay time dependence In the case of Fe-deficient plants, FC-R activities obtained with excised root tips were markedly ph-dependent, with values of and nmol Fe g 1 FW min 1 at ph values below and above 6.0, respectively (Fig. 1). A ph dependence was also found previously using whole roots of intact plants [19]. In excised root tips of Fe-deficient plants, Fig. 1. ph dependence of the FC-R in excised roots from Fe-deficient and Fe-sufficient sugar beet. The experiment was performed with the sugar beet NB1 NB4 hybrid, using MES (ph ) or TES (ph ) and a 10 min assay time. Data are means ± S.E. of five replications. Fig. 2. Time course of the FC-R-induced increase in absorbance per g FW of root tissue. Experiments were performed with the sugar beet Monohil hybrid. Data are means ± S.E. of 12 replications. A, at ph values 5.2 (5 mm MES) and 7.2 (5 mm TES); B, with nutrient solution plus 5 mm MES at ph 5.5 and 6.0; C, with 5 mm MES alone at ph 5.5 and 6.0.

3 Y.-C. Chang et al. / Plant Physiology and Biochemistry 41 (2003) Table 1 Effects of heavy metals during the assay on the functioning of the FC-R in excised roots from Fe-deficient sugar beet (in nmol Fe reduced g 1 FW min 1 ). The experiment was performed with the sugar beet Monohil hybrid. Data are means ± S.E. of nine replications. Values in the same column followed by the same letter are not significantly different (Duncan test at P 0.05) Plant Treatment Duration of incubation 15 min 30 min 60 min 120 min Fe-sufficient Control 4.9 ± 0.6 a 2.4 ± 0.3 a 5.0 ± 1.1 a 3.5 ± 0.5 a Fe-deficient Control 71.1 ± 8.9 b 76.3 ± 13.2 b 42.1 ± 3.3 bc 47.7 ± 11.6 b Fe-deficient CdCl 2 10 µm 69.0 ± 5.1 b 79.7 ± 10.5 b 46.1 ± 4.9 bc 36.5 ± 4.7 b Fe-deficient CdCl 2 50 µm 76.6 ± 10.9 b 82.3 ± 11.7 b 42.1 ± 4.6 bc 37.1 ± 6.0 b Fe-deficient Cd-EDTA 10 µm 71.5 ± 9.7 b 82.6 ± 10.7 b 43.7 ± 4.5 bc 52.6 ± 10.4 b Fe-deficient Cd-EDTA 50 µm 70.1 ± 8.3 b 89.4 ± 10.7 b 49.9 ± 2.9 c 55.6 ± 14.0 b Fe-deficient Pb-EDTA 1 mm 72.1 ± 6.8 b 82.6 ± 10.3 b 48.4 ± 3.4 bc 43.2 ± 7.1 b Fe-deficient Pb-EDTA 2 mm 57.8 ± 8.3 b 70.0 ± 14.7 b 35.9 ± 4.8 b 31.6 ± 7.3 b 2.2. Effects of Cd and Pb in the assay solutions on the activities of root FC-R activity FC-R activities were measured in the presence of different concentrations of CdCl 2, Cd-EDTA and Pb-EDTA in the assay solution. Since in short assay times (5 10 min) no effects of heavy metals were evident (data not shown), we used assay times in the range min, known to result in relatively low FC-R values in all plants. Iron-deficient roots not treated with heavy metals had FC-R activities eight to 30 fold higher (depending on assay time) than those found in Fe-sufficient controls (Table 1). The results showed that the presence of 10 and 50 µm of CdCl 2 or Cd-EDTA, and 1 mm Pb-EDTA did not significantly influence the FC-R activities of Fe-deficient plants, even when assay time was as long as 2h(Table 1). The only treatment causing some decreases in FC-R activity was 2 mm Pb-EDTA, which decreased enzyme activities by 10 33%, although the decreases were not statistically significant (Table 1). After min assay time, root FC-R values of Fe-deficient plants were generally in the range nmol Fe g 1 FW min 1. After 1 2 h of incubation, however, root FC-R activities of Fe-deficient plants decreased (both for plants treated or untreated with heavy metals) down to values of nmol Fe g 1 FW min 1, approximately 60% of those obtained at min. This was again caused by the non-linearity of absorbance increases with time. Chemical speciations indicated that most of the Fe (500 µm Fe-EDTA) added to the FC-R assay would stay in the form Fe-EDTA 1 (Table 2).Also, most of the Cd at 10 and 50 µm Cd-EDTA was predicted to stay as Cd-EDTA 2, and most of the Pb at 1 and 2 mm Pb-EDTA was predicted to stay as Pb-EDTA 2 (Table 2). With CdCl 2, Cd would be able to displace part of the Fe from Fe-EDTA, and the predicted equilibrium concentrations of free Cd 2+ would be approximately 1 and 15 µm with 10 and 50 µm CdCl 2 (Table 2). In this case, some Fe(II) BPDS could be formed spontaneously in the absence of root reducing activities, although concentrations of Fe-EDTA 1 would be still µm. Blank controls were always carried out in the absence of root tips to avoid these chemical-only, Fe-reduction unspecific effects Effect of short-term pre-treatment with Cd and Pb on root FC-R activity Plants were exposed to nutrient solutions containing Cd or Pb for 30 min and 1 h, heavy metals were then removed by extensive root washing with ultra-pure water and root tips were excised for the measurement of FC-R activity for 5 min as indicated in Section 4. Chemical speciation indicates that the major metal species during pre-treatment in the absence of Fe were free Cd 2+, Cd-EDTA 2 and Pb-EDTA 2 for CdCl 2, Cd-EDTA and Pb-EDTA treatments, respectively (Table 3). Free Cd 2+ concentrations were approximately 1, 2, 9 and 43 µm when using 10 µm Cd-EDTA, 50 µm Cd-EDTA, 10 µm CdCl 2 and 50 µm CdCl 2, respectively (Table 3). Iron deficient roots not treated with heavy metals showed higher FC-R activities (120 nmol Fe g 1 FW min 1 ) than Fe-sufficient controls (20 nmol Fe g 1 FW min 1 )(Fig. 3). After 30 min of treatment, root FC-R activities were decreased significantly by treating Fe-deficient plants with 50 µm Cd, but not with 10 µm Cd. With 50 µm Cd the most effective compound was CdCl 2, which decreased FC-R activity by 35%, whereas 50 µm Cd-EDTA only marginally Table 2 Metal speciation in the FC-R assay medium calculated with the software MinteqA2. Data are in µm Fe-EDTA Fe(OH)EDTA 2 4 Fe(II) BPDS 3 Cd 2+ Cd-EDTA 2 Cd(BPDS) 0 Pb-EDTA 2 PbHEDTA Pb 2+ Control CdCl 2 10 µm CdCl 2 50 µm Cd-EDTA 10 µm Cd-EDTA 50 µm Pb-EDTA 1 mm Pb-EDTA 2 mm

4 1002 Y.-C. Chang et al. / Plant Physiology and Biochemistry 41 (2003) Table 3 Metal speciation in the nutrient solutions containing 0 µm Fe calculated with the software MinteqA2. All concentrations are in µm Cd 2+ CdCl + 0 CdSO 4 0 CdHPO 4 Cd-EDTA 2 Pb-EDTA 2 Pb-HEDTA Pb 2+ CdCl 2 10 µm CdCl 2 50 µm Cd-EDTA 10 µm Cd-EDTA 50 µm Pb-EDTA 2 mm decreased FC-R activity. On the other hand, a 30 min treatment with 1 2 mm Pb-EDTA did not significantly change root FC-R activities. When the treatment lasted for 1 h, the effects of Cd and Pb on the FC-R activity of Fe-deficient plants were more marked (Fig. 3). FC-R activities had decreased, when compared to the control activity, by 50% (10 µm CdCl 2 ), 60% (50 µm CdCl 2 ), 17% (10 µm Cd-EDTA) and 34% (50 µm Cd- EDTA). Concentrations of 1 2 mm Pb-EDTA caused decreases in FC-R activities of approximately 30% Effect of long-term treatments with Cd and Pb on root FC-R activity Iron sufficient plants were exposed to nutrient solutions containing Cd or Pb for 7 10 d. Heavy metals were removed by extensive root washing with ultra-pure water and root tips were excised for the measurement of FC-R activity for 8 10 min as indicated above. Chemical speciation indicates that during the long-term treatment the concentration of Fe(III) EDTA was not affected at the concentrations of Cd and Pb used. The major Fe form was always Fe-EDTA 1, which was predicted to occur in concentrations of µm both in the absence and presence of Cd and Pb (Table 4).ForanygivenCdorPb treatment, both the major metal chemical species and the free metal concentrations were very similar in the presence or absence of Fe (Tables 3 and 4). Roots of sugar beet became dark when grown with Cd. Heavy metal treatments induced increases in the root tip FC-R activity of Fe-sufficient sugar beet (Fig. 4). In this experiment the FC-R activities of Fe-deficient and Fesufficient controls were approximately 130 and 25 nmol Fe g 1 FW min 1, respectively. Cadmium chloride induced larger increases in FC-R than Cd-EDTA, and for both Cd chemical forms the effects on FC-R were larger at 50 than at 10 µm concentrations. 3. Discussion Fig. 3. Effects of short-term heavy metal pre-treatments on the functioning of the root FC-R induced by Fe deficiency. Plants were grown with 45 µm Fe-EDTA (Fe-sufficient) or with 0 µm Fe (for Fe-deficient and different Cd and Pb treatments). Roots of Fe-deficient plants were excised after 30 and 60 min of exposure to heavy metals. Data are means ± S.E. of 6 9 replications. Columns marked with the same letter are not significantly different (Duncan test, P 0.05). Letters in italics are for the 60 min treatment. In this paper we describe different effects of the heavy metals Cd and Pb on the FC-R activity of excised roots. First, including up to 50 µm Cd and 2 mm Pb in the assay solution containing 500 µm Fe-EDTA did not cause changes in the root FC-R activities of Fe-deficient and Fe-sufficient plants even at very long assay times of 1 2 h. This indicates that the effects of relatively high concentrations of heavy metals on the reaction itself could be considered negligible, at least when assay times are kept short. In a previous study with cucumber, 250 µm Cd caused a significant decrease in FC-R function, whereas 250 µm Pb did not cause any effect [1]. Short-term pre-treatment (30 60 min) of intact Fedeficient plants with Cd and Pb in the absence of Fe induced significant decreases in their root FC-R activities. With Cd, Table 4 Metal speciation in the nutrient solutions containing 45 µm Fe calculated with the software MinteqA2. All concentrations are in µm Fe-EDTA Fe(OH)EDTA 2 + FeHPO 4 Cd 2+ Cd-EDTA 2 Pb-EDTA 2 PbHEDTA Pb 2+ Control CdCl 2 10 µm CdCl 2 50 µm Cd-EDTA 10 µm Cd-EDTA 50 µm Pb-EDTA 2 mm

5 Y.-C. Chang et al. / Plant Physiology and Biochemistry 41 (2003) Fig. 4. Elicitation of root FC-R activity by long-term heavy metal treatments. Roots were excised from Monohil sugar beet plants grown with 0 µm Fe (Fe-deficient), 45 µm Fe-EDTA (Fe-sufficient) or 45 µm Fe-EDTA with different heavy metal treatments. Data are means ± S.E. of 3 11 replications. Columns marked with the same letter are not significantly different (Duncan test, P 0.05). effects were more pronounced at 50 than at 10 µm, and they were stronger when Cd was added as CdCl 2 than when present in the form of Cd-EDTA. In a previous study with cucumber treated with heavy metals for 3 d, 5 µm Cd caused a 60% decrease in root FC-R activity of Fe-deficient plants, whereas 100 µm Pb did not cause any effect on the FC-R activity [1]. The heavy metal-induced decrease in the FC-R activity at short treatment times is unlikely to be caused by a direct interaction with the divalent metal plasma membrane transporters, since the reduced Fe(II) is sequestered during the assay by BPDS both in the presence or absence of heavy metals. Cadmium, however, can enter the root cell during the pre-treatment through the IRT-type Fe transporter [21] in turn inhibiting some cytoplasmic component controlling induced FC-R. The pathway for the entrance of Pb-EDTA is still unknown, but in plants such as Indian mustard the complex itself has been reported to reach the xylem [20]. Also, the FC-R may be directly affected if any plasma membrane redox component of FC-R is affected by the heavy metals or if binding sites are occupied by Cd or Pb in the absence of Fe. Long-term treatment of sugar beet plants grown in the presence of Fe with Cd and Pb caused a significant increase of the root FC-R activities. Increases in FC-R are expected, because Cd and Pb induce marked Fe deficiency symptoms in sugar beet, including leaf pigment concentration and photosynthesis decreases [12]. Increases in FC-R found with Cd and Pb, however, were not as large as those found in the total absence of Fe. This may be due to the moderation, due to the deleterious effects of both metals on root FC-R activities seen with short-term heavy-metal treatments, of the FC-R increase elicited by an induced Fe deficiency. Indeed, longterm Cd and Pb treatments led to increases in the metal concentrations in roots, which may reach values higher than 900 µg g 1 DW for both metals [12]. Chemical speciation indicate that the concentration of Fe(III) EDTA in the nutrient solution is not affected by heavy metals at the concentrations used. The effects on FC-R were more marked with CdCl 2 and at the highest concentration, when the predicted concentration of free Cd 2+ was approximately 43 µm, although increases in FC-R were also found in the other treatments, with free Cd 2+ concentrations in the range 1 8 µm. Results confirm, using the excised root technique proposed by Bavaresco et al. [3] and Moog et al. [15], that a large degree of induction of the FC-R activity occurs with Fe deficiency in roots of sugar beet. FC-R values found were approximately and nmol Fe g 1 FW min 1, for Fe-deficient and control plants, respectively, depending on experiment and genotype. When measured in intact plants, root FC-R activities of approximately 150 and 10 nmol Fe g 1 FW min 1 have been reported in Fe-deficient and Fe-sufficient Monohil sugar beet [19]. Also, FC-R values of approximately 138 and 13 nmol Fe g 1 FW min 1 have been reported previously in excised roots [13]. Therefore, in sugar beet the excised root technique may give values similar to those obtained with intact plants. These results are somewhat different from those found in tomato, where the excised root technique generally gives FC-R values twofold larger than those obtained with intact plants [25]. Results presented in this work give some information on how the excised root tip FC-R assays should be performed. The time used to perform the experiment is crucial, since after approximately 10 min the activity may not increase linearly. Values obtained depend on ph, although results were quite similar at ph Assays with or without nutrient solution give similar values. Also, performing controls to measure the reducing power of compounds excreted by root tips could be useful, since they may account for up to 10% of the total FC-R activities. In summary, results indicate that Cd and Pb affect FC-R activities in several different ways. Short-term treatments with these heavy metals induce a decrease in the FC-R activities of Fe-deficient plants, indicating that the enzyme is affected in some way by both metals. Long-term treatments, on the other hand, produce increases in the FC-R activities of Fe-sufficient sugar beet, confirming that they cause a heavy metal-induced Fe deficiency. These different effects should be taken into consideration when studying the effect of heavy metals on root FC-R activities. 4. Methods 4.1. Plant culture Sugar beet (Beta vulgaris L. Monohil hybrid from Hilleshög, Landskrona, Sweden, and NB1 NB4 hybrid from USDA, Salinas, CA, USA) was grown in a growth chamber with a photosynthetic photon flux density (PPFD) of 300 µmol m 2 s 1 photosynthetic active radiation (PAR) at a temperature of 25 C, 80% relative humidity and a photoperiod of 16 h light:8 h dark. Monohil sugar beet was used in most experiments, unless otherwise stated. Seeds were germinated and grown in vermiculite for 2 weeks. Seedlings

6 1004 Y.-C. Chang et al. / Plant Physiology and Biochemistry 41 (2003) were grown for two more weeks (30 plants in 45 l plastic boxes) in half-strength Hoagland nutrient solution with 45 µm Fe(III) EDTA. At this stage plants (thereafter called standard plants) were transplanted to 20 l plastic buckets (four plants per bucket) containing half-strength Hoagland nutrient solution [22] with either 0 or 45 µm Fe(III) EDTA. The ph of the Fe-free nutrient solutions was buffered at approximately 7.7 by adding 1 mm NaOH and 1 g l 1 CaCO Ferric chelate reductase measurements All experiments were conducted with excised root tips, following the technique described by Moog et al. [15] with some modifications. Sugar beet root tips (10 15 mm) were excised with a dissecting blade, placed in Eppendorf plastic tubes with 1 ml of assay solution and stirred on a shaker (Edmund Buhler SM, Germany), rotating at 180 rpm, in a darkroom at room temperature (22 24 C), for different time periods. The standard assay solution contained half-strength Hoagland solution (with micronutrients, excepting Fe), 5 mm MES (ph 6.0), 400 µm BPDS and 500 µm Fe(III) EDTA, unless otherwise stated. Measurements were also made in the absence of root tips, in triplicate, to correct for any unspecific Fe reduction. After developing the colour for a given time, the Eppendorf tube was vortexed for a few seconds to increase recovery in the solution of Fe(II) BPDS adhering to the roots. A volume of 0.8 ml of the assay solution was removed and placed in a new Eppendorf tube and absorbance was measured at 535 nm with a spectrophotometer (Shimadzu 2101 PC, Tokyo, Japan) using a 1 nmslit. After the assay, root tips were gently blotted on filter paper and weighed to determine root fresh mass. The ph dependence of the FC-R activities was measured with MES (ph ) or TES (ph ), in an experiment carried out with Fe-deficient and control root tips of the sugar beet NB1 NB4 hybrid. The dependence of FC-R on assay time was measured in two experiments: (a) with half-strength Hoagland solution and 5 mm MES ph 5.2 or 5 mm TES ph 7.2 at different reaction times (5, 10, 15 and 20 min) and (b) with half-strength Hoagland solution plus 5 mm MES or 5 mm MES alone at ph values 5.5 or 6.0 and at different reaction times (3, 5, 7, 10 and 15 min). The magnitude of Fe reduction caused by compounds released from the root tips to the assay solution was assessed at ph 5.5 and 7.0 by carrying out the incubation of root tips with assay solution without BPDS and Fe(III) EDTA, removing the root tips and then adding BPDS and Fe(III) EDTA Effects of Cd and Pb in the assay solutions on the functioning of FC-R Standard plants were placed in 20 l plastic buckets (four plants per bucket) and grown for 10 d with or without Fe. Root tips were excised and assayed (10 15 tips, approximately 6 12 mg FW in 1 ml assay solution) for FC-R activity at 15, 30, 60 and 120 min. The assay solution contained the components stated above, with or without different concentrations of CdCl 2, Cd-EDTA or Pb-EDTA Effects of short-term pre-treatments with Cd and Pb on the functioning of FC-R Standard plants were transplanted to 20 l plastic buckets (four plants per bucket) and grown for 10 d without Fe. Then Fe-deficient plants were placed in fresh half-strength Hoagland nutrient solutions (two plants per litre of solution in a black plastic container) containing 10 and 50 µm CdCl 2 or Cd-EDTA, or 1 and 2 mm Pb-EDTA for 30 min and 1 h. These short-term heavy metal pre-treatments were carried out in the growth chamber during the illumination period. After pre-treatment, roots were washed several times with MilliQ-quality H 2 O (Millipore) and root tips were excised. In this experiment FC-R activity was measured at ph 5.5 and for 5 min under otherwise standard conditions Effects of long-term treatment with Cd and Pb on the induction of FC-R Standard plants were transplanted to 20 l buckets (four plants per bucket) containing half-strength Hoagland nutrient solution with 45 µm Fe(III) EDTA and different concentrations of Cd or Pb. Treatments were 10 and 50 µm CdCl 2, 10 and 50 µm Cd-EDTA, 10 and 50 µm PbCl 2, and 10, 50, 500, 1000 and 2000 µm Pb-EDTA. Root tips were excised 7 10 d after imposing the heavy metal treatments. In this experiment 3 10 root tips per tube (0.5 1 cm, approximately 6 mg FW) were incubated with 1 ml assay medium in 10 mm MES, ph 6.0 and otherwise standard conditions (see above) for 8 10 min Chemical speciation Concentrations of the different ion species in the culture solutions were estimated with the software MinteqA2 (US Environmental Protection Agency, Washington, DC) [2], in order to assess metal (Fe, Pb and Cd) chemical species concentrations. Acknowledgements This work was supported by grants INCO-COPERNICUS PL and BOS from the Spanish MCYT to J. Abadía. Support to Y.C. Chang, Y. Gogorcena, M. Zouari and was provided by a post-doctoral fellowship from the Spanish Ministry of Education, Culture and Sports, a scientist contract from the Consejo Superior de Investigaciones científicas and a Master fellowship from the International Centre for Mediterranean Agronomic Studies, Agronomic Mediterranean Institute of Zaragoza, respectively. Authors gratefully acknowledge the technical assistance of Aurora Poc in growing the plants.

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