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Environmental and Experimental Botany 62 (2008) 316–322

Short and long-term uptake of Hg in white lupin plants:Kinetics and stress indicators

Elvira Esteban a,∗, Eduardo Moreno a, Jesus Penalosa a, Jose I. Cabrero a,Rocio Millan b, Pilar Zornoza a

a Departamento de Quımica Agrıcola, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spainb CIEMAT-DMA, Conservacion y Recuperacion de Suelos, Avenida Complutense 22, 28040 Madrid, Spain

Received 7 February 2007; received in revised form 20 July 2007; accepted 2 October 2007

bstract

The intrinsic characteristics of white lupin regarding biomass production and tolerance to abiotic stresses could make it a good candidate to besed in degraded mine soils containing mercury (Hg), but white lupin behaviour in response to Hg has to be previously evaluated. With this aim,inetic parameters of Hg uptake in short and long-term experiments, and Hg resistance of white lupin plants using several stress indicators weretudied. The plants were grown with increasing Hg doses in nutrient solutions (0, 5 and 10 �M). Hg uptake showed an active component in Hg influx,uggesting the existence of a low affinity root transporter that can be used for Hg uptake into white lupin root cells. Km and Vmax values obtainedor the saturable component were 217.7 ± 27.6 �M and 3.78 ± 0.18 �mol Hg g FW−1 h−1. Hg accumulation was concentration–time-dependent,howing a saturable behaviour for the lower doses but a linear behaviour for the highest ones. A high ability of Hg absorption by white lupin wasbserved both in short and long-term uptake experiments. The highest Hg dose reduced biomass production especially in the shoots. Moreover,ncreases in chlorophylls, malondialdehyde, total thiols and phenols were observed in Hg-stressed plants. The enhancement of total thiols andhenols levels in roots reduced oxidative stress for the 5 �M dose, but not for higher Hg levels. The deleterious effect of Hg was less marked inoot tissues, in spite of their accumulation of very high Hg amounts (99%) because of, at least in part, a combined increase in total thiols and

henolics able to minimize oxidative stress. Our results suggested that phenolic content in roots could be used as a new and easy-to-use indicatorf Hg presence. On the whole, white lupin showed a certain ability to survive in Hg-contaminated media and it would be possible to include it inombined decontamination strategies.

2007 Elsevier B.V. All rights reserved.

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eywords: Kinetic parameters; Mercury uptake; Stress indicators; Total pheno

. Introduction

Mercury (Hg) pollution is a global environmental problem,hose levels in soils have been increased nowadays due touman activities like mining of gold, silver and Hg itself, Cund Zn mining and smelting or coal burning. Mercury concentra-ions in the surface of non-contaminated soils ranged from 0.003

o 4.6 mg Hg kg−1 on a global scale (Steinnes, 1997). Althoughhe availability of soil Hg to plants is low, several studies havehown that plants accumulated Hg when they were exposed to

Abbreviations: DW, dry weight; FW, fresh weight; Hg, mercury; MDA, mal-ndialdehyde; MES, 2-[N-Morpholino]ethanesulphonic acid; -SH, total thiols;BA, 2-thiobarbituric acid; TCA, trichloroacetic acid.∗ Corresponding author. Tel.: +34 1 4974824; fax: +34 1 4973826.

E-mail address: elvira.esteban@uam.es (E. Esteban).

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098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2007.10.006

hite lupin (Lupinus albus L.)

g-contaminated soils. Mercury pollution of soils causes toxi-ity to plants and long-term effects on soil fertility (Wang andreger, 2004). Among other effects, Hg can induce inhibitionf plant growth, disturbances on water and nutrient uptake, pho-osynthesis, oxidative stress and enzymatic activities (Cho andark, 2000; Patra and Sharma, 2000; Ortega-Villasante et al.,005).

Mercury accumulation in plants has been studied in severallant species, like pea and spearmint (Beauford et al., 1977),pring wheat, sugar beet, oil-seed rape, white clover and willowGreger et al., 2005), rice (Du et al., 2005), tomato (Cho and Park,000) and alfalfa (Ortega-Villasante et al., 2005). Hg distribution

n wild plant species has also been studied (Millan et al., 2006;

oreno-Jimenez et al., 2006). Plants are able to take up Hg fromhe soil or nutrient solution but, generally, absorbed Hg is slightlyranslocated to shoots. Nevertheless, kinetic parameters of Hg2+

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ptake in higher plants have not been reported, although sometudies with the cyanobacterium Nostoc calcicola Breb. haveeen carried out (Pandey and Singh, 1993).

On the other hand, mine soils are generally low in nutrientsnd organic matter. Therefore, the inclusion of nitrogen-fixinglants in the stabilizing vegetation should improve ecosystemevelopment by naturally increasing the nitrogen content in theoil, and promoting the maintenance of the plant cover (Frerott al., 2006). White lupin has shown a relatively high toleranceo several contaminants (Zornoza et al., 2002; Vazquez et al.,005) and its efficiency in phytostabilisation for those elementsVazquez et al., 2006), but no information is available for Hg.ata from soils belonging to the Almaden mining district (Spain)

anged from 5 to 1750 mg Hg kg−1 (Millan et al., 2006). In ordero include white lupin in degraded mine soils of the Almadenrea for improving ecosystem development and reducing Hgovement out of this polluted area, its response to Hg has to be

valuated. In this sense, kinetic parameters of Hg2+ uptake andong-term Hg accumulation, distribution and evaluation of Hgesistance for white lupin have been studied under controlledonditions. Oxidative stress, total thiols, chlorophylls and phe-ols were used as biomarkers of the degree of Hg stress sufferedy white lupin.

. Materials and methods

.1. Plant growth

White lupin seeds (Lupinus albus L.) cv. Marta wereurface-sterilised in 10% (v/v) sodium hypochlorite for5 min, rinsed thoroughly with deionised water and germi-ated in darkness at 28 ◦C for 3 days on water-moistenedlter paper. The seedlings obtained were placed in plas-

ic containers (8 L) with half strength, continuously aeratedutrient solution: 1.5 mM Ca(NO3)2; 4.0 mM KNO3; 1.5 mMH2PO4 and 1.0 mM MgSO4. Micronutrients were sup-lied as: 36 �M Fe-EDDHA; 33 �M MnSO4·H2O; 1.6 �MnSO4·7H2O; 1.6 �M CuSO4·5H2O; 46 �M H3BO3 and.1 �M (NH4)6Mo7O24·4H2O. The pH of the nutrient solutionanged from 5.5 to 6.0.

.2. Short-term Hg2+ uptake: kinetics parameters

Seedlings were grown for 2 weeks in 3 L containers ingrowth cabinet with the following conditions: night/day18/21 ◦C, photoperiod 8/16 h, relative humidity of 80%,

hotosynthetic photon flux density of 1300 �mol m−2 s−1.xperimental design used for Hg uptake was similar to thene described by Esteban et al. (2003). Four replicate sam-les of whole roots (excised at the node) were used. Forptimisation of incubation time, roots were incubated inerated solution containing 0.5 mM CaCl2, 2 mM MES (2-[N-orpholino]ethanesulphonic acid) and 50 �M Hg at pH 6.0

uring 10, 20, 30, 60 or 100 min. For the kinetic studies, rootsere incubated in aerated solution containing 0.5 mM CaCl2,mM MES (2-[N-Morpholino]ethanesulphonic acid) and 0, 10,0, 50, 100, 200, 500 or 1000 �M Hg at pH 6.0 for 20 min both

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rimental Botany 62 (2008) 316–322 317

t 25 ◦C and ice-cold (<2 ◦C) conditions. After root incubation,lant material was rinsed in a fresh ice-cold solution containingmM CaCl2 and 5 mM MES at pH 6.0 for 2 min and incubated

n a fresh ice-cold nutrient solution of the same compositionuring 15 min for desorbing out no-uptaken Hg. Fresh weightsf the roots were determined before analysis.

.3. Long-term Hg uptake

Hg treatments were initiated 10 days after sowing. Plantsere grown in a glasshouse under the following environmental

onditions: night/day T 12–28 ◦C, relative humidity of 50–80%nd a photosynthetic photon flux density of 500 �mol m−2 s−1.g was added as HgCl2 to the nutrient solutions to give con-

entrations of 0, 5 and 10 �M Hg. Deionised water was used forreparing all solutions and it was added to replace losses fromranspiration every 2 days. The entire nutrient solutions werehanged weekly and sampled. Three replicates of each treatmentere established in a randomised block design. Plants were har-ested 1, 2, 3 and 4 weeks after the initiation of Hg treatments.t harvest, roots and shoots from each plant were separated and

heir fresh weight recorded. One thousand milligrams (FW) oflant material were frozen in liquid N2 and stored at −20 ◦C.he remaining plant matter was dried at room temperature tovoid Hg losses for 1 week, until constant, weight to determinery weights.

.4. Mercury analysis

Nutrient solutions were analysed directly. Hg uptake wasetermined by its disappearance of the nutrient solution aftereplacing water loss due to transpiration. An Advanced Mercurynalyser (AMA-254, LECO Company) measured Hg in nutri-

nt solutions. Five replicates of each sample were measured andhe results are expressed as the mean value within two standardeviations.

Plant material (200 mg DW shoots and roots for long-termxperiment and whole fresh roots for short-term experiments)as digested with a mixture containing HNO3:H2O2:H2O

3:2:10, v:v:v) for 30 min at 125 ◦C under 1.5 kPa of pres-ure (Lozano-Rodrıguez et al., 1995). Hg in these samples waseasured by ICP-MS. Evaluation of Hg losses during sam-

le processing was carried out using spikes with 2 mg Hg L−1,nd at least 97% recovery was obtained, with a coefficient ofariance of 4.18%. Certified reference material BCR-CRM 62olive leaves; 0.28 ± 0.02 mg Hg kg−1) was also mineralised and.27 ± 0.01 mg Hg kg−1 was obtained, with a coefficient of vari-nce of 3.90% (Moreno-Jimenez et al., 2006).

.5. Stress indicators

Frozen samples were homogenised to a fine powder in liquid2 with an ice-cooler mortar and pestle. Such pulverised mate-

ial was used to determine chlorophyll, lipid peroxides and totalhiols. Chlorophyll was extracted with 80% (v/v) acetone. Thebsorbance of the acetone extracts at 645 and 663 nm were deter-ined and the chlorophyll a and b contents calculated (Wellburn,

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Concentration dependent uptake kinetics for Hg2+ influx wassignificantly affected by temperature. At 20 ◦C, Hg2+ influxwas graphically resolved into a saturable (hyperbolic) com-

18 E. Esteban et al. / Environmental and

994). The amount of lipid peroxides was determined as mal-ndyaldehyde (a citotoxic product of lipid peroxidation that isormally considered as the major 2-thiobarbituric acid react-ng compound) as has been described (Lozano-Rodrıguez et al.,997). Plant material (100 mg FW) was extracted in 2.0 mL ofCA–TBA–HCl reagent (15% (w/v) TCA; 0.37% (w/v) TBAnd 0.25 mM HCl). The extract was heated in a sand bath (90 ◦C,0 min). After cooling, the flocculent precipitate was removedy centrifugation at 11,000 × g for 10 min. Absorbance of theupernatant was measured at 535 nm and corrected for non-pecific turbidity by subtracting the absorbance at 600 nm. Totalhiols were assayed using 100 mg FW of plant material with.4 mL of NaOH (1 M) containing NaBH4 (1 mg mL−1) and.2 mL of de-ionised water. After centrifugation (11,000 × g,0 min), 0.5 mL of supernatant was added to 0.5 mL of 5,5′-ithiobis(2-nitrobenzoic acid) dissolved in neutralizing buffer0.5 M potassium phosphate, pH 7.2). Absorbance was measuredt 410 nm (Jocelyn, 1987). Total phenols were extracted from50 mg DW roots with 10 mL of acidulated methanol 0.1% HClnder 200 rpm (4× 10 mL, each time shaking during 4 h, at roomemperature). After filtration, the supernatants were combinednd adjusted to 100 mL with methanol. Total phenol contentas determined by the method adapted from Singleton and Rossi

1965) using the Folin–Ciocalteu reagent. To each tube, 0.50 mLf the extract was added followed by 10 mL of distilled waternd 0.50 mL of Folin–Ciocalteu reagent. After 3 min, 10 mLf 20% sodium carbonate was added. The tubes were capped,ixed thoroughly and heated at 40 ◦C, 40 min. The blue col-

ration was read at 685 nm against a blanck standard. Resultsere expressed in mg of gallic acid g−1 DW of roots.

.6. Calculations and statistical analyses

Effective accumulation in whole plant and in shoot, translo-ation and the ratio of Hg content in shoot and root after Hgreatments were calculated as:

Effective accumulationwhole plant (%)

= Total Hg in plants (mg)

Total added Hg (mg)× 100 (1)

Effective accumulationshoot (%)

= Total Hg in plants (mg)

Total added Hg (mg)× 100 (2)

Translocation (%)

= Total Hg accumulated in whole plant (�g)

Total Hg accumulated in shoots (�g)× 100 (3)

Hg concentration in shoot and root ratio (%)

= Hg concentration in shoot (�g)

Hg concentration in root (�g)× 100 (4)

Background concentrations of Hg in plants grown withoutg were subtracted from concentrations in the treated plantsrior to the calculations. The background concentrations of Hg

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rimental Botany 62 (2008) 316–322

n plants were due to the materials used for cultivation and theg contamination in air. A linear relationship between absorbedg versus Hg added to the plants (R2 = 0.95; P < 0.001), and alsoetween total Hg accumulated by the plant versus absorbed HgR2 = 0.99; P < 0.001) was obtained.

Michaelis–Menten functions were fitted to the concentration-ependent influx data using SigmaPlot 9.0 software to calculatehe kinetic parameters. Values in the tables and figures are

eans ± standard errors (S.E.) of three independent replicates.ne-way ANOVA was used to compare the results obtained with

he different Hg treatments, followed by a post hoc multiple com-arisons of means using the Duncan test (P < 0.05). Significantifferences among the Hg treatments are expressed by differentetters (a, b, c) in the tables and figures. All calculations were

ade using SPSS v. 12.0 software.

. Results

.1. Kinetic parameters of Hg2+ uptake in short-termxperiments

Time course of Hg2+ accumulation is shown in Fig. 1. Hg con-entration in roots increased for the first 100 min, but the increaseas more rapid for the first 30 min, significantly decreasing the

ate of Hg uptake for longer periods of time (28 and 67%, respec-ively for 60 and 100 min). A linear increase was clearly observedor the first 30 min. These results have been obtained after a5 min desorption time in 5 mM CaCl2, to ensure desorptionf most of the Hg2+ bound in the apoplast. Similar desorptionimes and Ca concentrations have been used for Zn2+ and Cd2+

Lasat et al., 1996; Lombi et al., 2001; Zhao et al., 2002). So,ncubation times of 20 min have been used for the concentration-ependent incubation experiments. This incubation time has alsoeen used in many experiments dealing with the kinetics of ionptake (Lasat et al., 1996; Cohen et al., 1998; Lombi et al., 2001;

ig. 1. Hg uptake by roots of L. albus at different incubation times in short-termxperiments. Vertical bars indicate (±) standard error of the mean (n = 3).

E. Esteban et al. / Environmental and Experimental Botany 62 (2008) 316–322 319

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ig. 2. Hg uptake by roots of L. albus at different temperatures in short-termxperiments: 20 ◦C (open circles) or ice-cold (<2 ◦C, full circles). Vertical barsndicate (±) standard error of the mean (n = 3).

onent and a non-saturable linear component (Fig. 2), whilenly a non-saturable linear component was best fitted for thece-cold Hg influx. The saturable component was more clearlybserved for the lower Hg external concentrations, while theinear component was more evident at the highest Hg concen-rations. A significant reduction of Hg influx was observed inhe ice-cold experiments, where metabolic energy-dependentptake processes should be minimal. So the ice-cold influxould be attributed to apoplastic adsorption of Hg that remainedound to the cell walls after desorption, while Hg influx at0 ◦C should include an active component in Hg influx, sug-esting the existence of a root transporter that can be usedor Hg uptake into root cells. When subtracting influx databtained for 20 ◦C and ice-cold experiments, the contribu-ion of the saturable component will only remain. These data

ere best fitted to a hyperbola (Fig. 3), with Km and Vmaxalues for the saturable component of 217.7 ± 27.6 �M and.78 ± 0.18 �mol Hg g FW−1 h−1.

ig. 3. Saturable component of Hg uptake by roots of L. albus in short-termxperiments. Data were obtained by subtracting Hg influx data at 20 ◦C andce-cold. Vertical bars indicate (±) standard error of the mean (n = 3).

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ig. 4. Effect of Hg supply on Hg accumulation in long-term experiments: (a)hoots and (b) roots. Vertical bars indicate (±) standard error of the mean (n = 3).

.2. Hg2+ accumulation and distribution in long-termxperiments

Hg concentrations in plant parts increased significantly withncreasing Hg dose and exposure time (Fig. 4a and b). Theelationship between shoot and root Hg concentrations andxternal Hg level added to the plants followed a saturationrend both in shoots and in roots (Fig. 4a and b, P < 0.001).t the lower Hg doses supplied (<5 �mol Hg per plant),

he relationship between plant concentration and externaloncentration followed a Michaelis–Menten kinetics, butor the highest Hg doses a sharp increase in shoot and rootg concentrations was obtained, so that a linear component

ssociated to the hyperbola was the best fit found. The externalg supply to reach one half of the maximum plant concentration

nd the maximum plant Hg concentration obtained for theaturable component were, respectively, 1.4 ± 0.2 �mol plant−1

nd 10.2 ± 1.0 �mol Hg g DW−1 in roots; and.2 ± 0.4 �mol plant−1 and 45.7 ± 5.6 �mol Hg kg DW−1

n shoots, respectively.

Several parameters of Hg accumulation in plant parts are

isplayed in Table 1. Only 30–40% of the Hg applied wasccumulated in the plant, and around 0.16–0.25% was found

320 E. Esteban et al. / Environmental and Experimental Botany 62 (2008) 316–322

Table 1Mercury accumulation in whole plants or in shoot and relation of Hg concen-tration between shoot and root in 28-day-old Hg-treated white lupin plants

Hg doses

5 �M 10 �M

Effective accumulationwhole plant

(%)39.90 ± 0.59a 32.29 ± 0.35b

Effective accumulationshoot (%) 0.25 ± 0.01a 0.16 ± 0.01b

Translocation of Hg (%) 0.62 ± 0.02a 0.48 ± 0.02b

Hg concentrations in shoot androot ratio (%)

0.54 ± 0.03a 0.46 ± 0.02b

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Table 3Effect of Hg supply on MDA, -SH, chlorophyll and phenol concentrations in28-day-old white lupin plants

Plant part Hg doses

0 �M 5 �M 10 �M

MDA (nmol g−1 FW)Shoots 25.9 ± 0.6a 28.7 ± 0.8b 30.2 ± 1.1b

Roots 45.4 ± 0.9a 48.9 ± 1.2a 56.2 ± 1.4b

Total thiols (nmol g−1 FW)Shoots 184.8 ± 3.5a 224.9 ± 3.7b 244.2 ± 4.7b

Roots 48.0 ± 0.7a 79.9 ± 1.0b 99.2 ± 1.7c

Total chlorophyll (mg g−1 FW)Shoots 0.94 ± 0.02a 1.15 ± 0.04b 1.18 ± 0.02b

Total phenols (mg g−1 DW)Roots 2.23 ± 0.11a 3.61 ± 0.13b 4.80 ± 0.14c

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oncentration of Hg in control plants is subtracted. Data are means ± S.E.,n = 3). Means within a row followed by the same letter do not differ significantlyn Duncan test (P < 0.05).

n shoots. A small part of the Hg taken up is translocated tohe shoot (0.48–0.62%) and the shoot/root Hg concentrationatio (expressed as percentage) was around 0.50%. For all thebove-mentioned parameters, values are significantly lower forhe 10 �M Hg dose than for the 5 �M Hg dose.

.3. Plant growth and water content of 28-day-old plants

The most obvious symptom of Hg toxicity was the inhibitionf plant growth. Compared to the control plants, the 10 �M Hg-reated plants showed clear reductions in shoot length (19%),hoot weight (29%) and root weight (22%). For the 5 �M Hgose, the shoot length (10%) and weight (20%), and the fresheight of the root (12%) were also significantly decreased.owever, Hg-treated and untreated plants showed no significantifferences in terms of root length. Differences between the 5 and0 �M Hg-treated plants were only observed for the shoots. Inhe 10 �M treatment, the roots turned a brownish colour. Waterontent of the Hg-treated shoots was significantly lower thanhat of the untreated shoots, although no significant differencesn the water content were seen between the shoots from the 5

nd 10 �M Hg-treated plants. Significant, Hg dose dependentecreases were seen in the water content of Hg-treated rootsTable 2).

able 2ffect of Hg supply on length, weight and water content of 28-day-old white

upin plants

lant part Hg doses

0 �M 5 �M 10 �M

ength (cm plant−1)Shoots 17.70 ± 0.20a 15.93 ± 0.24b 14.37 ± 0.12c

Roots 38.27 ± 0.40a 38.27 ± 0.40a 36.87 ± 0.13a

resh weight (g plant−1)Shoots 7.66 ± 0.21a 6.10 ± 0.11b 5.45 ± 0.17c

Roots 8.13 ± 0.19a 7.14 ± 0.30b 6.35 ± 0.25b

ater content (g 100 g−1)Shoots 86.60 ± 0.12a 85.88 ± 0.09b 85.59 ± 0.07b

Roots 92.77 ± 0.25a 90.46 ± 0.42b 89.14 ± 0.55c

ata are means ± S.E., (n = 3). Means in a row followed by the same letter doot differ significantly in Duncan test (P < 0.05).

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ata are means ± S.E., (n = 3). Means within a row followed by the same lettero not differ significantly in Duncan test (P < 0.05).

.4. Stress indicators

Significant, Hg dose dependent increases were seen inhe total thiol and phenol concentrations of Hg-treated roots,mounting similar values for both stress indicators (1.6 timesn the 5 �M treatment and 2.1 times in the 10 �M treatment).ompared to the control plants, the levels of MDA (10%: 5 �Mnd 16%: 10 �M), total -SH (22%: 5 �M and 32%: 10 �M) andhlorophylls (22%: 5 �M and 25%: 10 �M), were significantlyncreased in the shoots of plants grown with Hg; however, noignificant differences were observed between the two Hg doses.n the roots, the levels of MDA increased 1.2 times for the 10 �Mg treated compared to untreated roots, whereas no significantifferences were obtained between roots from the control and�M Hg-treated plants (Table 3).

. Discussion

Because of low Hg available to plants in normally occurringcosystems or conditions, information is meagre about itsptake and metabolism in higher plants. It is generally acceptedhat plant uptake of toxic ions like Hg2+ might take place viahe same uptake processes used for essential micronutrientons (Patra and Sharma, 2000). Hg2+ preferentially binds withulphur- and nitrogen-rich ligands as amino acids (Nieboernd Richardson, 1980). Hg2+ uptake by white lupin plantsas studied in both short and long-term experiments. In the

hort-term experiments, an active component on Hg influxas found (Fig. 3). To the best of our knowledge, no previous

nformation about the kinetic parameters of Hg uptake in plantsas been reported. Pandey and Singh (1993) described the Hg2+

ptake pattern of the cyanobacterium Nostoc cacicola Breb.,nding also an active component in Hg uptake, with Km 1.0 �Mnd Vmax 0.21 nmol mg protein−1 min−1. Ion antagonism with

u2+ and synergism with Ni2+ suggested that Hg uptake was

aking place through one or various essential nutrient membraneransporters, as is also probably the case in plant roots. Byxamining the various essential nutrients as grouped by their

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E. Esteban et al. / Environmental and

hemical properties in the periodic table, we can identifyhe closest chemical relatives of Hg as Zn, Cu or Fe, so thatheir transporters could be possible candidates to facilitate Hgnflux. Km and Vmax values obtained for Hg are significantlyigher than those described for Zn or Cd in Thlaspi sp., peand wheat (Cohen et al., 1998; Hart et al., 1998; Lombi et al.,001). Calcium channels, as observed for Cd in the Prayonhlaspi ecotypes (Zhao et al., 2002), or even aquaporines,ater channels that seem to be blocked by Hg (Johansson et al.,000), could also partly contribute to this high Hg uptake abilityf white lupin. Also, white lupin is able to develop strategies inrder to increase local iron and phosphorus availability in soilsNeumann and Martinoia, 2002). These characteristics modify,or example, the down-regulation of the phosphate/arsenateptake by phosphate (Esteban et al., 2003). In a similar way, Feptake ability might be naturally modified and this mechanismight also contribute to Hg uptake by white lupin roots. In

ny case, the results obtained indicate a low affinity of theransporter for Hg uptake, but a high capacity of white lupino uptake Hg, as a very high Hg concentration is necessary toaturate the transporter. An only system seems to be operatingor all the range of concentrations tested. In this work, a wide Hgoncentration range has been studied, in order to test root uptakeoth for high and low Hg concentrations, although many ofhese Hg concentrations will never be found in soil conditions.

Long-term Hg uptake in this study can reflect and be influ-nced by a number of factors, including Hg compartmentationn roots, as well as Hg translocation and use in shoots. Hg con-entration in plants over 28-days of metal exposure increasedith increasing amounts of Hg added. The pattern of long-termg accumulation in shoots and roots can be dissected into lin-

ar and hyperbolic (saturable) components (Fig. 4a and b). Thisattern resembled the results obtained for short-term kinetics,uggesting a saturation of the Hg uptake ability of the planthen Hg supply is increased. In order to reach 50% saturationf Hg uptake capacity both in root and shoot around of 1.4 �molg per plant had to be added to the growth media.Our study further showed that white lupin efficiently accu-

ulates Hg in hydroponics. Thus, Hg concentration of rootseached 1700 and 3052 mg Hg kg−1 DW for plants grown in 5nd 10 �M, respectively. Concerning effective accumulation ofg in white lupin (Table 1), the values obtained reached sim-

lar percentages than those found in pea, wheat and rape byreger et al. (2005). The accumulation of Hg in shoots of white

upin was much lower than in the roots, whereas only a smallmount of the total Hg absorbed was allocated to the shoots0.62–0.48%; Table 1). This has also been observed for otherlant species (Beauford et al., 1977; Cavallini et al., 1999; Patrand Sharma, 2000; Wang and Greger, 2004; Greger et al., 2005;oreno-Jimenez et al., 2006). Such a low translocation of Hg to

he shoots is probably due to white lupin roots having a strongffinity for Hg, whereby most of the Hg in solution could berapped in the root tissues mainly bound to the root cell walls,

s suggested earlier (Cavallini et al., 1999; Wang and Greger,004).

Mercury produced a reduction in the growth of white lupinlants. This effect was mainly observed in the shoot, while roots

aatH

rimental Botany 62 (2008) 316–322 321

ere apparently only damaged by the highest Hg dose appliedTable 2). Indeed, no visible symptoms of toxicity were seenn the 5 �M Hg-treated plants at all. The inhibition of growthnd reductions in biomass production are phenomena frequentlybserved in plants exposed to toxic levels of Hg (Patra andharma, 2000). Since the roots are probably the first to suf-er Hg injury, the inhibition of root weight was also seen inlants treated with the 5 �M Hg dose. The lower sensitivity ofhite lupin roots to Hg could be explained by a capacity of thelant to accumulate the metal in a non-active form, as observedn willow clones by Wang and Greger (2004) and in wheat byavallini et al. (1999). Moreover, Hg caused disturbances inater balance judging by the observed reduction in plant water

ontent, probably by inhibiting the absorption and transloca-ion of water (Barcelo and Poschenrieder, 1990). According toohansson et al. (2000) HgCl2 can inhibit water flow, mainlyhrough the blockage of water channels.

In most cases, Hg exposure promotes reductions in chloro-hyll levels. Conversely, the leaves of white lupin grown underg exposure (5 �M and/or 10 �M) gave higher levels than non-g treated leaves (Table 3). Patra et al. (2004) reported that Hg

ncreased the levels of chlorophylls and carotenoids at shorterxposure times, but decreased them at prolonged exposure time.esides, Hg produced an enhancement of MDA concentrations

n white lupin shoots for both Hg doses, but only in the rootsf plants grown with the highest dose (Table 3). MDA is anndex of lipid peroxidation and, therefore, of oxidative stress.ncreases of MDA by Hg exposure have been observed in otherases (Shaw, 1995; Cho and Park, 2000; Ortega-Villasante et al.,005; Moreno-Jimenez et al., 2006).

Other stress indicators were enhanced by Hg presence in thistudy (Table 3). Thus, a strong increase of total thiols and phe-olics has been found in Hg treated roots, doubling basal levelsn those treated with the highest Hg dose. Metal concentration inhe roots was significantly (P < 0.001) and positively correlatedith -SH and phenol contents (r = 0.83 and 0.89, respectively).henolic compounds are plant carbon-based secondary metabo-

ites able to protect plant tissues from oxidative damage becausef their antioxidant capacity, and their accumulation in plantss stimulated by various biotic and abiotic stresses (Dixon andaiva, 1995). The increase of these compounds could be aefence mechanism developed by the roots of white lupin to min-mize the oxidative damage caused by Hg exposure and reflectedy an enhanced MDA production (Table 3). Thiol groups playn important role in the cytoplasmic detoxification mechanismgainst heavy metals, but are also required to counteract all theeleterious effect of oxidative stress (Noctor and Foller, 1998).he increase in thiolic and phenolic levels observed in the rootsf white lupin plant under Hg exposure could be a combinedtrategy to reduce the oxidative stress caused by Hg.

In conclusion, white lupin plant has shown a high ability ofg uptake, as well as a low translocation rate to the shoot. Theinetic parameters of Hg influx showed the contribution of an

ctive component, with Km and Vmax values of 217.7 ± 27.6 �Mnd 3.78 ± 0.18 �mol Hg g FW−1 h−1 respectively. These are,o the best of our knowledge, the first kinetic parameters ofg uptake reported for plants. Toxicity symptoms were only

3 Expe

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cnotecps

A

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R

B

B

C

C

C

D

D

E

F

G

H

J

J

L

L

L

L

M

M

N

N

N

O

P

P

P

S

S

S

V

V

W

W

Z

22 E. Esteban et al. / Environmental and

bserved for the 10 �M Hg dose, as reflected by the reductionn weight and the presence of oxidative stress. The combinedncrease in total thiolic and phenolic in roots could act as arotective mechanism to minimize oxidative stress in responseo Hg to some extent. To our knowledge, the closer relationshipetween total phenolics and Hg exposure has not been reportedefore. Total phenolic contents in roots could be used as anndicator of Hg stress in white lupin.

Overall, the results support the hypothesis that white lupinould be used in the combined strategies to recover Hg contami-ated soils. The use of white lupin could associate the advantagesf using leguminous plants and the efficiency in phytostabilisa-ion shown for other trace elements (Vazquez et al., 2006). Itsfficiency in accumulating Hg in the roots, with very low translo-ation rate to the shoots, could make white lupin a good choice inhytostabilisation, preventing leaking of Hg from contaminatedoils.

cknowledgements

The Spanish Ministry of Education and Science, projectsTM2004-06715-CO2-01 and CTM2005-04809/TECNO,nancially supported this research. The authors are grateful to.J. Sierra (CIEMAT) and to I. Rivas (SIDI-UAM) for the Hg

eterminations.

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