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Published in J. Environ. Qual. 33:1271-1279 (2004).
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TECHNICAL REPORTS

Heavy Metals in the Environment

Heavy Metal Accumulation by the Halophyte Species Mediterranean Saltbush

Stanley Luttsa,*, Isabelle Lefèvrea, Christine Delpéréea, Sandrine Kivitsa, Caroline Dechampsa, Antonio Robledob and Enrique Correalc

a Unité de Biologie végétale, Institut des Sciences de la Vie, Université catholique de Louvain, 5 (Bte13) Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium
b Thader-consultoria Ambiental, C/Poeta Sánchez Marigal 8, 300004 Murcia, Spain
c Consejería de Agricultura, Agua y Medio Ambiente, Centro de Investigación y Desarollo Agroalimentario, 30150 La Alberca, Murcia, Spain

* Corresponding author (lutts{at}bota.ucl.ac.be).

Received for publication April 8, 2003.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify Cd- and Zn-accumulating plants exhibiting a high growth rate, seeds from the halophyte species Mediterranean saltbush (Atriplex halimus L.) were collected on a heavy-metal-contaminated site in southeastern Spain (Llano del Beal, Cartagena). Seedlings from this ecotype were exposed for 3 wk to 0.1 mM Cd or Zn in a nutrient solution in a fully controlled environment. All plants remained alive and no significant growth inhibition was recorded until the end of the experiment. Mean Cd and Zn accumulation in aerial parts was 830 and 440 mg kg–1, respectively, and the rate of metal translocation even increased with the duration of stress exposure. Resistance to heavy metals in this species may be partly linked to precipitation of Cd in oxalate crystals in the stems. A Cd-induced decrease in glutathione concentration also suggests that phytochelatins overproduction may occur in these conditions. We conclude that Mediterranean saltbush, which is able to produce up to 5 Mg dry matter ha–1 yr–1, may be an effective species for phytoextraction and should be tested for this purpose in field conditions.

Abbreviations: GSH, reduced glutathione • GSSG, oxidized glutathione • RGR, relative growth rate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEAVY METALS and metalloids are an increasing environmental problem worldwide. Some industrial activities and agricultural practices increase their level in the substrate, and the possible introduction of these elements in the food chain is an increasing human health concern (Cakmak et al., 2000). Engineering industrial techniques may efficiently be used to clean up contaminated soils but most of them require sophisticated technology and are therefore expensive and suitable only for small, polluted areas (Moffat, 1995). Phytoextraction implies the use of plants to remove pollutants from the environment and has been proposed as an interesting alternative solution for the decontamination of large areas (Cunningham and Berti, 1993; Brooks, 1998). Most studies dealing with phytoextraction focus on hyperaccumulating plants able to concentrate high levels of heavy metals in their aerial parts without showing any symptom of injury. The strategies of resistance in those plants involve several mechanisms such as the vacuolar sequestration of heavy metals linked to overproduced organic acids (malate, citrate, or oxalate) or phytochelatins produced from glutathione (Salt et al., 1998). Heavy-metal-tolerant plants belonging to the genus Alyssum, Thlaspi, or Silene have been identified for a long time (Brooks, 1998) and their use for phytoextraction purposes has been recommended (Schwartz et al., 2003; Zhao et al., 2003). Most hyperaccumulators, however, are difficult to manage and have a shallow root system, and their interest is therefore limited in the case of deep contamination (Keller et al., 2003).

The use of deep-rooting halophyte species is of particular interest in this context because these plants are naturally present in environments characterized by an excess of toxic ions, mainly sodium and chloride. Several studies demonstrated that some tolerance mechanisms operating at the whole-plant level are not always specific to sodium and that other toxic elements such as copper, zinc, or cadmium may accumulate in salt glands or trichomes in tamaris [Tamarix aphylla (L.) Karst.], marsh-daisy [Armeria maritima (Mill.) Willd.], and gray mangrove [Avicennia marina (Forsk.) Vierh.] (Hagemeyer and Waisel, 1988; Neumann et al., 1995; McFarlane and Burchett, 1999). Among the halophyte flora, species belonging to the genus Atriplex may be of special interest because of their high biomass production associated with a deep root system able to cope with the poor structure and xeric characteristics of several polluted substrates. These species also naturally produce high amounts of oxalic acid, which may assume positive functions in tolerance mechanisms to heavy metal stress (van Baelen et al., 1980; Mazen and El Maghraby, 1997; Sayer and Gadd, 2001). Fourwing saltbush [Atriplex canescens (Pursh) Nutt.)] has been especially recommended for revegetation of mine sites and other harsh environments (Baumgartner et al., 2000; Glenn et al., 2001; Newman and Redente, 2001). Other species, such as Gardner's saltbush [A. gardneri (Moq.) D. Dietr.] (Salo et al., 1996), Australian saltbush (A. semibaccata R. Br.) (de Villiers et al., 1995), shadscale saltbush [A. confertifolia (Torr. & Frem.) S. Wats.] (Wood et al., 1995), and Suckley's endolepis [A. suckleyi (Torr.) Rydb.] (Voorhees et al., 1991) have been successfully tested for revegetation purposes. Atriplex species are able to accumulate high amounts of Se (Vickerman et al., 2002) and some of them have been shown to accumulate B (Watson et al., 1994) or Mo (Voorhees et al., 1991). To the best of our knowledge, no data are available concerning Zn and Cd accumulation in these species.

The first step to assess the potential interest of a plant species for phytoextraction is to quantify, in a fully controlled environment, the mean level of toxic metal accumulation in relation to the growth rate. The present study deals with Zn and Cd, two elements sharing numerous similar chemical properties that are often present concomitantly in polluted areas. Accumulations of these elements were quantified in the Mediterranean halophyte species Mediterranean saltbush, which is present as a natural invading shrub in several mining areas of northern Africa and southern Europe. Plants were exposed to a nutrient solution with similar levels of Zn or Cd to determine the specific toxicity of these elements in relation to their level of accumulation. The internal plant concentration of oxalate and glutathione was also determined to gain information on the mechanisms of tolerance to heavy metals and thus to define valuable criteria to select metal accumulating individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Growth Conditions
Seeds of Mediterranean saltbush were collected during autumn 2000 from plants growing on a mining site located at Llano del Beal, near Cartagena, in southeastern Spain (37°32' N, 0°56' W). The opencast mining activity in this area stopped in 1987 and has given rise to the creation of quarries, slag heaps, and ponds where vegetation colonization is slow. Mediterranean saltbush was found colonizing the surface of a tailing where the mean plant cover was 50%. The soil texture was 22.1% sand, 55.4% fine and coarse silt, and 22.5% clay. Its organic matter concentration was 1.7% and pH (H2O) and pH (KCl) were 6.7 and 6.2, respectively. The concentrations of nutrients and heavy metals in soil were (in mg kg–1 dry matter): 4809 Ca, 123 K, 31 Na, 384 Mg, 12 P, 3965 Zn, 12.5 Cr, 6.8 Ni, 65 Cu, 12.7 Cd, and 5671 Pb. Seeds collected from separate plants were pooled. After removal of the bracts, seeds were germinated in plastic jars filled with a substrate of 18.8% silt, 37.3% clay, and 42.1% sand, having a bulk density of 1.6 g cm–3, a gravimetric water content at field capacity of 8%, and an electrical conductivity of 2.08 dS m–1, with a pH (KCl) of 7.2 and a total percentage of humus of 1.9%. The mineral concentration was estimated as (in mg kg–1 dry matter): 741 K, 390 P, 1782 Mg, 71 Na, 14012 Ca, 4.1 Zn, and 0.2 Cd. The jars were incubated in a growth chamber under a 12-h photoperiod (mean light intensity = 110 µmol m–2 s–1) provided by Osram Sylvania (Danvers, MA) fluorescent tubes (F36W/133-T8/CW) with day and night temperatures of 28 and 20°C, respectively. Substrate and young seedlings were sprayed daily during a few minutes with sterile deionized water. After 3 wk, corresponding to the four-leaves stage, young seedlings were transferred to polyethylene pots (12 x 12 x 9 cm) containing the same solid substrate in a greenhouse under natural lighting supplemented by Phillips (Eindhoven, the Netherlands) lamps (G/93/2; 400 W) providing a minimal photon flux density of 175 µmol m–2 s–1, a temperature of 25 ± 2°C, and a relative humidity of 70%. After 3 wk, plants were adapted to a nutrient solution [Table 1; modified Hoagland, according to Gulick and Dvorák (1987)] in phytotrons (light intensity = 175 µmol m–2 s–1 supplied by Sylvania tubes [F96T12 CW/VHO], constant temperature of 25 ± 2°C, relative humidity of 70%) and fixed on a polystyrene plate floating at the surface of 2-L plastic tanks (nine plants per tank). After 1 wk of acclimation in the absence of stress, ZnSO4 or CdCl2 were added to reach a concentration of 0.1 mM. The amount of solution in each tank was readjusted daily and solutions were renewed every week.


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  		Table 1. Basal nutrient solution used for culture of Mediterranean saltbush (after Gulick and Dvorák, 1987).

 
Determination of Growth Parameters and Metal Concentration
For each treatment, five plants were randomly harvested after 1, 2, and 3 wk of exposure to heavy metal stress. Roots were quickly rinsed during 30 s in 0.1 mM SrCl2 and then gently blotted dry. Roots, stems, and leaves of each plant were separated and weighed for fresh weight determination. Dry weight was determined after 2 d of incubation in an oven at 70°C. Water content was calculated for each organ as [(fresh weight – dry weight)/fresh weight] x 100.

Mean relative growth rates (RGR) were determined separately for roots, stems, and leaves, for each treatment and each week of exposure to stress according to the following formula: RGR = (ln DW2 – ln DW1)/{Delta}t, where DW1 and DW2 are the mean dry weights at the beginning and at the end of the time interval, respectively and {Delta}t is the time interval.

For each sample, digestion of dry matter (approximately 50 mg) was accomplished at 80°C in 35% (v/v) HNO3 (Zhao et al., 1994). Minerals were dissolved in 0.1 M HCl and element concentrations (cations and P) were determined using an inductively coupled argon plasma emission spectrophotometer (JY48; Jobin Yvon, Edison, NJ) calibrated with certified cadmium and zinc nitrate standard solutions (Merck, Darmstadt, Germany; concentrations confirmed by complexometric titration). All measurements were performed in three replicates.

Mean rates of element transport from roots to shoots were calculated for each element and separately for each week of exposure to stress according to Salim and Pitman (1983) as: J = [(M2 – M1)/(R2 – R1)] x (ln R2 – ln R1)/{Delta}t, where M1 and M2 are the amounts of elements in shoots (leaves + stems) at the beginning and end of the considered period of exposure to stress, R1 and R2 are the corresponding root dry weights, and {Delta}t is the time interval.

Oxalate and Glutathione Quantification
Oxalate and glutathione concentrations were determined after 1, 2, and 3 wk in roots, leaves, and stems on three samples per treatment, each sample containing the organs of four pooled individual plants.

Extraction of total and water-soluble oxalate was performed according to Karimi and Ungar (1986) with slight modifications. Total oxalate was extracted by heating a mixture of ground material and 0.25 M HCl (1:100 w/v) at 70°C during 1 h. A 5-mL sample of the extract was precipitated overnight at 4°C after addition of 1 mL of precipitating reagent (96.5 g anhydrous sodium acetate in 250 mL water + 18 g anhydrous calcium acetate in 250 mL of 50% acetic acid). The precipitate was dissolved in 5 mL of washing reagent (240 mL of 96% ethanol + 125 mL of concentrated ammonium hydroxide), ovendried at 100°C, and finally dissolved in 5 mL of 2 M H2SO4. Quantification of oxalate was performed after complete oxidation and reaction of the resulting H2O2 with 3-methyl-2 benzothiamine hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB) using a Sigma Diagnostics kit (Sigma Aldrich, St. Louis, MO) according to the manufacturer's instruction. The concentration of indamine issued from this reaction was determined by reading the optical density at 590 nm.

For the extraction of water-soluble oxalate, a mixture of ground plant material and deionized water (1:100 w/v) was heated at 70°C for 1 h and filtered. The same procedure was repeated three times. To 10 mL of the extract, 0.12 mL of concentrated HCl were added and the combined extract was again heated at 70°C for 20 min. Any precipitate was removed by centrifugation. Water-soluble oxalate was precipitated and quantified as for total oxalate. Acid-soluble oxalate was determined by subtracting water-soluble from total oxalate.

Reduced glutathione (GSH) was assayed by the enzymatic recycling procedure in which it is sequentially oxidized by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and reduced by nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of glutathione reductase according to Griffith (1980). The ground tissue (approximately 1 g fresh wt.) was suspended in 4 mL 5% sulfosalicyclic acid and centrifuged at 10000 x g for 10 min. A 330-µL aliquot was removed and neutralized by addition of 18 µL 7.5 M triethanolamine. One 150-µL sample was then used to determine concentrations of GSH plus oxidized glutathione (GSSG). Another was pretreated with 3 µL 2-vinylpyridine for 60 min at 20°C to mask the GSH by derivatization and to allow the subsequent determination of GSSG alone. In each case, 50-µL aliquots of the two types of sample were mixed with 700 µL of 0.3 mM NADPH, 100 µL of DTNB, 150 µL of buffer containing 125 mM sodium phosphate, and 6.3 mM EDTA (pH 6.5). A 10-µL aliquot of glutathione reductase (EC 1.6.4.2; 5 U mL–1; 1 unit will reduce 1 µmol oxidized glutathione min–1 at pH 7.6 at 25°C) was then added and the change in absorbance at 412 nm monitored at 30°C. A standard curve was prepared using solutions of GSH and GSSG.

Statistical Treatment
Two independent experiments were performed and exhibited similar trend. For each treatment (stressing agent x duration of stress), three tanks containing nine plants each were used in a randomized complete block design. The metal concentrations in plant tissues were log-transformed to normalize the frequency distribution. Percentage data (i.e., water content and percentage of insoluble oxalate) were transformed to arcsine values before statistical analysis. Data obtained for each type of organ were analyzed by a two-way analysis of variance (ANOVA, stressing agent and duration of stress as levels of classification) performed with the model procedure of SAS Version 6.12 (SAS Institute, 1997). If the F value indicated significant differences (P < 0.05), mean differences were compared according to the Scheffé F test for growth parameters, glutathione and oxalate concentrations, and according to the Waller–Duncan k ratio t test for metal concentration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Heavy Metal on Plant Growth
Heavy metal stress had no effect on plant survival and all plants remained alive until the end of the treatment. After 1, 2, and 3 wk, the mean number of leaves per plant was 12.3, 15.6, and 17.2 and treatment had no effect on this parameter. The RGR values progressively decreased with time in control plants. The mean relative growth rates of roots, stems, and leaves were not affected by zinc treatment compared with controls (Fig. 1), while RGR values decreased for stems and leaves of cadmium-treated plants for the first two weeks of exposure to stress. In stems, such a decrease was associated with a decrease of 18% in the mean length of the internodes but not with a decrease in the number of nodes. It is noteworthy that RGR during the third week of treatment was much higher in Cd-exposed plants than in controls, suggesting that these plants recovered after an initial shock and that growth was delayed rather than irreversibly reduced. As a consequence, the final dry matter values of roots (2.6 ± 0.3 g) and leaves (7.2 ± 0.4 g) harvested on Cd-treated plants were statistically similar to the control (P = 0.61 and 0.72 for roots and leaves) and only stems exhibited a significant reduction in the mean dry matter (3.1 ± 0.2 g) compared with control plants (4.7 ± 0.6 g) (P < 0.03).



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  		Fig. 1. Relative growth rates (RGR) of roots, stems, and leaves of Mediterranean saltbush exposed during 1, 2, and 3 wk to either 0.1 mM Cd or Zn. The RGR values were estimated on a dry weight basis for the first (0->1), second (1->2), and third (2->3) week of exposure to heavy metal stress.

 
Neither Zn nor Cd treatment had any effect on the water content of roots and leaves (mean values of 92.3 and 84.2%, respectively), suggesting that metal toxicity did not induce secondary water stress, at least for the duration of exposure considered in the present study.

Heavy Metal Accumulation and Mineral Nutrition
Accumulation of cadmium in Cd-treated plants already occurred after 1 wk of exposure (Fig. 2) and increased until the end of the treatment without exhibiting any saturation trend. Although Cd accumulation was higher in roots, a significant amount of this element accumulated in the aerial parts. After 3 wk of exposure to 0.1 mM Cd, Cd constituted 3174, 1151, and 618 mg kg–1 dry matter in roots, stems, and leaves, respectively. The rates of translocation of Cd from roots to shoots even increased with the duration of stress exposure (Table 2).



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  		Fig. 2. Cadmium concentration (in mg kg–1 dry matter) in roots, stems, and leaves of Mediterranean saltbush exposed for 1, 2, and 3 wk to 0.1 mM Cd in nutrient solution. Each value is the mean of five replicates and vertical bars are standard errors. Note that the vertical scaling is not the same for roots and aerial parts.

 

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  		Table 2. Rates of transport (J) of Cd and Zn from roots to shoots in plants exposed to 0.1 mM Cd or Zn during 3 wk.{dagger}

 
Zinc accumulation in response to Zn treatment follows the same pattern (Fig. 3). Zinc concentrations were higher in the roots than in the aerial parts. Zinc, however, progressively accumulated in both stems and leaves; after 3 wk of exposure, Zn concentrations were 1423, 431, and 460 mg kg–1 dry matter in roots, stems, and leaves, respectively. While the rate of zinc transport remained constant in control plants, it increased in Zn-exposed plants (Table 2).



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  		Fig. 3. Zinc concentration (in mg kg–1 dry matter) in roots, stems, and leaves of Mediterranean saltbush exposed for 1, 2, and 3 wk to 0.1 mM Zn in nutrient solution. Each value is the mean of five replicates and vertical bars are standard errors. Note that the vertical scaling is not the same for roots and aerial parts.

 
Cadmium treatment increased Zn concentrations by more than 20% in all plant organs when recorded at the end of the stress period (279, 175, and 136 mg kg–1 dry matter in roots, stems, and leaves of control; 370, 201, and 175 mg kg–1 dry matter in roots, stems, and leaves of Cd-treated plants); however, considering the heterogeneity of the plant material, such increase in Zn concentrations was only significant at the leaf level (P < 0.05).

As far as other elements are concerned (Table 3), Cd and Zn had contrasting effects. No effect on K and Mg concentrations was recorded in response to heavy metals. Zinc treatment significantly decreased calcium concentration in all plant organs after 21 d of treatment. Sodium was quantitatively important in this halophyte species, even in control conditions, and it increased in the leaves in a highly significant way (P < 0.01) in response to both Cd and Zn treatment. Cadmium treatment strongly increased P in all organs (P < 0.001) while Zn treatment increased Fe concentration in roots but decreased it in stems. Zinc treatment strongly increased Mn concentration in roots but had no effect on Mn concentration in the aerial parts.


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  		Table 3. Effect of exposure to heavy metals (0.1 mM Cd or Zn) during 3 wk on K, P, Mg, Ca, Na, Fe, and Mn concentrations in roots, stems, and leaves of the halophyte species Mediterranean saltbush.{dagger}

 
Oxalate and Glutathione Concentrations
Total oxalate concentration (Fig. 4) was lowest in roots, intermediate in stems, and highest in leaves. In all organs, total oxalate increased in response to Cd; such an increase was significant after 2 and 3 wk in roots and was already observed after 1 wk of exposure to 0.1 mM Cd in both stems and leaves. Oxalate accumulation was typically lower in response to Zn compared with Cd, except for roots exposed for 2 wk to heavy metal. Whatever the treatment, almost all oxalate found in roots was in the soluble form. Percentages of insoluble oxalate (Table 4) never exceeded 20% in leaves and were not significantly modified by the treatment. In contrast, a consistent proportion of oxalate in stems was in the insoluble form. At the end of the stress exposure, percentages of insoluble oxalate were significantly higher in Cd- and Zn-treated plants than in control plants.



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  		Fig. 4. Total oxalate concentration (in µmol g–1 fresh matter) in roots, stems, and leaves of Mediterranean saltbush exposed for 1, 2, and 3 wk to either 0.1 mM Cd or Zn. Each value is the mean of three replicates and vertical bars are standard errors.

 

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  		Table 4. Percentage of insoluble oxalate in roots, stems, and leaves of Mediterranean saltbush exposed for 1, 2, or 3 wk to 0.1 mM Cd or Zn.{dagger}

 
Both GSH and GSSG concentrations in leaves and GSSG concentration in stems decreased with the age of the control plants (Fig. 5) and GSH concentrations were always higher than GSSG concentrations, whatever the treatment, analyzed organ, or duration of stress exposure. Zinc had no significant effect on glutathione concentration (P > 0.3). Although Cd did not affect root GSH or GSSG concentration, it drastically reduced the endogenous GSH concentration in leaves and GSSG concentration in both stems and leaves after only 1 wk of exposure to stress. The mean difference between the control and Cd-treated plants, however, decreased with the duration of exposure and after 3 wk of stress, such a difference was still significant for GSSG concentration in the leaves only (P < 0.01).



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  		Fig. 5. Reduced (GSH) and oxidized glutathione (GSSG) (in nmol g–1 fresh matter) in stems and leaves of Mediterranean saltbush exposed for 1, 2, and 3 wk to 0.1 mM Cd in nutrient solution. Each value is the mean of three replicates and vertical bars are standard errors. Note that vertical scaling is not the same for GSH and GSSG.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Can Mediterranean Saltbush be Used for Phytoextraction?
The present work shows that Mediterranean saltbush is tolerant to both Cd and Zn and that it may accumulate these elements in the aerial part without showing any significant decrease in terms of biomass production during a 3-wk exposure to high concentrations in nutrient solution.

According to Cunningham and Berti (1993), plants may be suitable for phytoextraction purposes if they contain more than 10000 mg toxic elements per kg of dry matter. The putative interest of a given species, however, depends on the best quantitative compromise between metal concentration and biomass production. Plants are considered as hyperaccumulators if they contain more than 10000 mg kg–1 Zn or 100 mg kg–1 Cd and if the shoot to root ratio for heavy metal concentration is greater than 1 (Brooks, 1998). The usual level of biomass production of Mediterranean saltbush in natural environments is around 5 Mg dry matter ha–1 yr–1, which corresponds to a mean yield of more than 30 Mg fresh matter ha–1 yr–1 (Kelley et al., 1982; Eldin, 1993). Proper management of the culture by application of fertilizers and chemicals and increase of the density of plants in the field should lead to an increase of biomass production by a factor of 3 (Lailhacar et al., 1995; Bouzid and Papanastasis, 1996). A biomass production comprised between 2 and 5 Mg ha–1 yr–1 has been reported for the hyperaccumulator Thlaspi caerulescens J. Presl & C. Presl (Robinson et al., 1998). If we assume that growth of Mediterranean saltbush is not inhibited by the external dose of Zn and Cd used in this study, and considering that plants accumulate 830 mg kg–1 dry matter Cd and 440 mg kg–1 dry matter Zn (taking into account the proportions of leaves and stems at the time of harvest), one may expect to remove 4.15 kg ha–1 Cd and 2.2 kg ha–1 Zn per year for a basal biomass production of 5 Mg dry matter ha–1 yr–1. The potential of Cd tolerance is of particular interest if we assume that the dose of Cd used in the present work exceeds by far the value found in soil solution, even for highly polluted substrate. It is also obvious that the above-reported value corresponds to a theoretical value quantified in nutritive solution and that removal of Cd from a soil would be expected to be substantially lower as uptake largely depends on the availability of Cd in polluted substrates. On another hand, it has to be mentioned that the dose of Zn used in the present work is very low compared with the doses used in other studies or present on contaminated soils (Robinson et al., 1998; Keller et al., 2003; Schwartz et al., 2003; Zhao et al., 2003). Since we intended to compare the specific effects of Cd and Zn on plant behavior, we decided to use similar doses for both elements. Therefore, the data for Zn extraction potential may be underestimated considering the low level of Zn used in our work.

Further experiments are necessary to check that there is no growth inhibition on a long-term basis and that the rates of metal uptake and translocation are maintained at subsequent developmental stages. Finally, parameters influencing the bioavailability of heavy metals in soil conditions also have to be tested and potential removal rates should be confirmed by experiments using soil as a substrate for plant growth.

Effect of Cadmium and Zinc on Mineral Nutrition and Water Status
Mediterranean saltbush is a hardy xero-halophyte species that is able to cope with long periods of drought with high salinity levels. Drought resistance mechanisms in Mediterranean saltbush may indirectly contribute to Cd and Zn tolerance, since high levels of heavy metals are responsible for secondary water stress in plants (Poschenrieder et al., 1989). It may thus be able to acclimate on heavy-metal-contaminated mine soils, which are often characterized by poor structure. Heavy metal stress also had a limited effect on the concentration of other elements. A decrease in calcium may be partly explained by the fact that divalent cations, such as Cd2+, can translocate in the plant through nonselective calcium channels (Clemens et al., 1998). Such a process may have occurred in our material but internal Ca concentration remained well above the limit of deficiency considered for higher plants. Fodor et al. (1996) demonstrated that in cucumber (Cucumis sativus L.), a strong inhibition of photosynthesis may result from iron deficiency in Cd-treated plants: while 10 µM Cd reduced by more than 90% the leaf Fe concentration of cucumber, 0.1 mM Cd had no effect at all on the leaf iron concentration of Mediterranean saltbush. Zinc did not inhibit iron uptake but it reduced its translocation from roots to shoots; it also reduced iron concentration in the stems but not in the leaves. The increase of Na concentrations in leaves in response to Cd and Zn is a surprising result: Mediterranean saltbush is a halophyte species but the nutrient solution used in the present experiment had a low electrical conductivity and should not be considered saline. A specific increase in Na concentrations was found in Mediterranean saltbush exposed to drought in the absence of salt (Martínez et al., 2003). This suggests that Na may play a specific role in response to abiotic stress, which may be the case for other C4 species, which need Na for efficient phosphoenolpyruvate regeneration.

It has been demonstrated that Mediterranean saltbush is able to accumulate high amounts of Na in trichomes covering the leaf surface but that other elements may also accumulate when present in excess (Freitas and Breckle, 1993). Trichomes accumulate both Cd and Zn in the resistant species Arabidopsis halleri (L.) O'Kane & Al Shehbaz (Küpper et al., 2000) and Cd in the halophyte species Athel tamarisk [Tamarix aphylla (L.) Karst.] (Hagemeyer and Waisel, 1988). Specific overexpression of a gene coding for a metallothionein (MT2) has been reported in trichomes of various species (Foley and Singh, 1994; García-Hernandez et al., 1998), thus suggesting that they constitute important sites for toxic metal accumulation.

Possible Involvement of Oxalate and Glutathione in Resistance of Mediterranean Saltbush to Heavy Metals
Coprecipitation of Cd and/or Zn with Ca in oxalate structures may be another efficient way of detoxification, as most Atriplex species produce high amounts of oxalic acid (Karimi and Ungar, 1986). Such a process contributes indeed to Cd resistance in water hyacinth [Eichhornia crassipes (Mart.) Solms] (Mazen and El Maghraby, 1997) and oxalic acid is also able to bind Zn and form insoluble crystals (Sayer and Gadd, 2001). Calcium and cadmium have a similar ionic radius and share several physical properties, which may explain why Cd is often detected in raphids or crystals of calcium oxalate (van Baelen et al., 1980). Our results support the hypothesis that Cd precipitation in the form of oxalate in shoots may occur in Mediterranean saltbush. Indeed, there is a significant increase of total oxalate in response to Cd stress (Fig. 4) and the proportion of insoluble oxalate was higher in stems (Table 4), where we found the largest concentration of Cd (Fig. 2). These findings suggest that precipitation may occur in stems to prevent toxic Cd from reaching the photosynthetically active leaves. The situation is less clear for Zn, although Zn treatment also slightly increased the proportion of insoluble oxalate. The process probably did not occur in roots, where only a small amount of total oxalate was found, nor in leaves, where almost all oxalate remained in the soluble form.

Beside oxalate, glutathione is another important compound that may be involved in resistance to heavy metals. As an endogenous antioxidant molecule, it helps to reduce the effect of secondary oxidative stress resulting from the production of reactive oxygen species (Noctor et al., 2002) but it also constitutes the precursor of phytochelatins, which are small peptides binding to Cd and accumulating in vacuoles (Cobbett and Goldsbrough, 2002). Although glutathione has been found to increase in response to numerous environmental stresses in several species (Noctor et al., 2002, and references therein), our results reveal that Cd induced a decrease of both the reduced and the oxidized form of glutathione in the shoots of Mediterranean saltbush. Further quantification of endogenous phytochelatins should help us to determine as to whether the observed decrease in GSH is related to overproduction of these metal-binding peptides, since Cd stress has been shown to stimulate {gamma}-Glu-Cys dipeptidyl transpeptidase (EC 2.3.2.15) involved in the last step of phytochelatin synthesis (Cobbett and Goldsbrough, 2002). The fact that GSSG also decreased in shoots suggests that stress may simultaneously activate glutathione reductase involved in recycling of GSSG through GSH. The decrease in the endogenous glutathione pool, however, did not appear to affect the survival or the growth of the tested plants. Detoxification of Cd-induced reactive oxygen species and Cd precipitation through oxalate may be complementary ways to overcome cadmium toxicity.

From the present data, it may be concluded that Mediterranean saltbush behaves as a Cd accumulating tolerant species that holds promise for phytoextraction purposes. This species, however, should still be tested in the field under metal-contaminated conditions. Further work is needed to obtain information about the putative heavy metal tolerance mechanisms of this species.


   ACKNOWLEDGMENTS
 
This work was supported by European Union (Convention no. ERB IC 18-CT98-0390) and Fonds National de la Recherche Scientifique (FNRS-FRFC; Convention no. 2.4565.02). The authors are very grateful to Mr. J.S. Sironi for his kind help in collecting the seeds and soil samples from field conditions and to Mr. L. Gerlache from the Unité des Eaux et Forêt (UCL) for his valuable technical assistance during mineral analysis.


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