Fractionation of Stable Zinc Isotopes in the Field-Grown Zinc

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Fractionation of Stable Zinc Isotopes in the Field-Grown Zinc Hyperaccumulator Noccaea caerulescens and the Zinc-Tolerant Plant Silene vulgaris Ye-Tao Tang,†,‡,§ Christophe Cloquet,∥ Thibault Sterckeman,*,§ Guillaume Echevarria,§ Jean Carignan,⊥ Rong-Liang Qiu,†,‡ and Jean-Louis Morel§ †

School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275, Peopleʼs Republic of China Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou 510275, Peopleʼs Republic of China § Laboratoire Sols et Environnement, INRA, Université de Lorraine, BP 172, 2 avenue de la forêt de Haye, F-54505 Vandoeuvre-lès-Nancy Cedex, France ∥ CRPG-CNRS, Université de Lorraine, 15 rue Notre-Dame-des-Pauvres BP 20, 54501 Vandoeuvre-lès-Nancy, France ⊥ Takuvik, CNRS-ULaval, Québec G1 V 0A6, Canada ‡

S Supporting Information *

ABSTRACT: Stable Zn isotope signatures offer a potential tool for tracing Zn uptake and transfer mechanisms within plant−soil systems. Zinc isotopic compositions were determined in the Zn hyperaccumulator Noccaea caerulescens collected at a Zn-contaminated site (Viviez), a serpentine site (Vosges), and a noncontaminated site (Sainte Eulalie) in France. Meanwhile, a Zn-tolerant plant (Silene vulgaris) was also collected at Viviez for comparison. While δ66Zn was substantially differentiated among N. caerulescens from the three localities, they all exhibited an enrichment in heavy Zn isotopes of 0.40−0.72‰ from soil to root, followed by a depletion in heavy Zn from root to shoot (−0.10 to −0.50‰). The enrichment of heavy Zn in roots is ascribed to the transport systems responsible for Zn absorption into root symplast and root-to-shoot translocation, while the depletion in heavy Zn in shoots is likely to be mediated by a diffusive process and an efficient translocation driven by energy-required transporters (e.g., NcHMA4). The mass balance yielded a bulk Zn isotopic composition between plant and soil (Δ66Znplant−soil) of −0.01‰ to 0.63‰ in N. caerulescens, indicative of high- and/or low-affinity transport systems operating in the three ecotypes. In S. vulgaris, however, there was no significant isotope fractionation between whole plant and rhizosphere soil and between root and shoot, suggesting that this species appears to have a particular Zn homeostasis. We confirm that quantifying stable Zn isotopes is useful for understanding Zn accumulation mechanisms in plants.

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shoots without showing visual toxicity symptoms.6,7 Compared to the nonaccumulating species Thlaspi arvense L., N. caerulescens exhibits higher rates of Zn uptake,8 more efficient translocation of Zn from root to shoot,9 and stronger Zn sequestration in the shoots as well.10,11 High uptake rates are suggested to involve a high-affinity transport system mediated by the ZIP (zinc/iron-regulated transporter protein) family,12 while efficient xylem loading of Zn is thought to be driven by

INTRODUCTION

Zinc is an essential trace element for higher plants and almost all living organisms, but it can be highly toxic at elevated concentrations. Zinc concentration in plants above 300 μg·g−1 is normally considered as toxic.1 However, Zn-hyperaccumulating plant species can accumulate and tolerate much higher levels of Zn in their tissues.2 One of the best-known Zn hyperaccumulators is Noccaea caerulescens (J. & C. Presl) F. K. Mey (formerly Thlaspi caerulescens), which has been reported to have a potential for cleaning up trace-metal-contaminated soils.3−5 Intensive studies have been performed regarding mechanisms of Zn accumulation and tolerance in N. caerulescens. This species can accumulate up to 3% (dry weight) of Zn in its © 2012 American Chemical Society

Received: Revised: Accepted: Published: 9972

April 16, 2012 August 9, 2012 August 14, 2012 August 14, 2012 dx.doi.org/10.1021/es3015056 | Environ. Sci. Technol. 2012, 46, 9972−9979

Environmental Science & Technology

Article

Silene vulgaris (Moench) Garcke. S. vulgaris was chosen as it also colonizes on the Zn-contaminated (Viviez) site around N. caerulescens, and it adopts a different Zn tolerance mechanism in comparison with N. caerulescens. The results are expected to provide an understanding of Zn cycling and metabolism during absorption, translocation, and sequestration processes in N. caerulescens.

an upregulation of heavy-metal-transporting ATPase (HMA4).13 However, so far most physiological and biochemical studies have been conducted only with indirect measurements, that is, artificially spiking Zn or radioactive Zn experiments, and fundamentally require further confirmation by direct techniques for measuring interactions between intact plants and substrates under natural conditions.14 In this case, a high-precision multicollector inductively coupled plasma mass spectrometer (MC-ICP-MS) provides a powerful tool with which to study metal cycling in soil−plant systems, by documenting isotopic variations.15 Generally, isotope fractionation occurs during the uptake and transport of a metal within plants, and any variations in stable isotopic composition observed can create an isotope “fingerprint” of the plant in question. This then contributes to our understanding of metal dynamics in soil− plant systems. A pioneer study by Weiss et al.16 demonstrated that Zn isotopic fractionation occurs during Zn absorption and translocation in rice, tomato, and lettuce grown in hydroponics. The enrichment in heavy Zn isotopes of 0.08−0.18‰ from solution to root and the depletion in heavy Zn of −0.13 to −0.26‰ from root to shoot, indicate a preferential adsorption of heavier isotopes on the root surface and a membranetransport-mediated uptake. However, soil-grown rice tends to enrich heavy Zn isotopes in its shoot relative to plant-available Zn, pointing to a critical role of phytosiderophore in Zn complexation and uptake from Zn-deficient soil.14 In a pristine watershed of Cameroon, the roots and shoots of six plant species were enriched in heavy Zn isotopes, while only tree leaves were depleted in heavy isotopes by up to −0.91‰.17 A linear correlation was proposed between plant length and the extent of Zn fractionation,17 which has been confirmed in fieldgrown bamboos.18 Recently, Caldelas et al.19 suggested that isotopic fractionation differed substantially in a Zn-tolerant species, Phragmites australis, exposed to contrasting Zn concentration in solution. In conditions of Zn sufficiency (3.2 μM), transporter proteins (e.g., ZNT1) would facilitate Zn uptake for plant Zn requirement, while in conditions of excess Zn (2 mM), complexation with ligands and compartmentation of Zn in nonactive parts are likely to retain Zn in roots and protect the plant from toxicity. On the basis of these observations, Jouvin et al.20 proposed a conceptual model and highlighted the role of several dominant processes (e.g., speciation in media, adsorption on root cell walls, and uptake pathways) that would lead to Zn isotope fractionation during uptake and translocation. The above studies indicate that the extent of Zn isotopic fractionation appears to be species-specific and Zn uptake and transport can be conditioned by Zn levels and speciation in media and plant physiological status. As far as we know, however, little effort has been devoted to the topic of Zn isotopic composition in hyperaccumulating plants. Only recently, Aucour et al.21 demonstrated that Zn isotopic fractionation differs between the Zn hyperaccumulator Arabidopsis halleri and the nonaccumulator Arabidopsis petraea grown hydroponically and may reflect the extent of root to shoot translocation. Therefore, the objectives of this study were to characterize the Zn isotopic fractionation associated with Zn transfer in the Zn hyperaccumulator N. caerulescens grown naturally in three different soil-plant systems (a Zn-contaminated site, a noncontaminated site, and a serpentine site) and to compare this fractionation with that of the Zn-tolerant species

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MATERIALS AND METHODS Sampling. During May and June 2011, field investigation was performed in a Zn-polluted site (Viviez), a typical serpentine site (Vosges), and a nonmetallicolous site (Sainte Eulalie). The sampling site of Viviez (N 44°33.57′, E 02°13.02′; 350 m altitude) is at a roadside 200 m from a former Zn/Cd smelter operated from late in the nineteenth century until the end of the 1980s. The soils here have consequently been heavily contaminated with zinc, cadmium, lead, copper, and arsenic around adjacent railway yards and the roadsides for more than a kilometer in some directions.22 At Vosges (N 47°54.50′, E 06°57.50′; 715 m altitude), samples were collected on an ultramafic outcrop of Bergenbach in the municipality of Oderen and Fellering, located in northeast France. This soil has developed on ultramafic bedrock, which is a highly serpentinized peridotite known as harzburgite.23 The site of Sainte Eulalie (N 44°48.21′, E 04°12.78′; 1270 m altitude) is located in the Massif Central in France and is a permanent mountainous pasture laid on a granitic bedrock. To investigate the Zn isotopic fractionation within soil−plant systems, complete plants of N. caerulescens and their rhizospheric soils were sampled at their fruiting stage in each site (one plant and soil in Viviez, two in Sainte Eulalie, and one in Vosges). The rhizospheric soil, which is considered as that adhering to the roots and within the space explored by the roots,24 was taken from the root surface by digging up the root from the soil with a small shovel and shaking the whole root system. Generally the plant roots grew alone at Viviez and Vosges sites, which are metallicolous soils composed of many gravels and sands. Thus it is relatively easy to acquire the complete root. At Sainte Eulalie site which represents a clay soil, the roots intertwined with some other grass roots that require more attention. Therefore here a roughly 15 × 15 × 15 cm soil column for each plant was dug and taken back to lab for separation of the root. The complete plants include roots, stems, and leaves. During the sampling period, the plants were at fruiting stages and some plant parts (e.g., flowers and seeds) have already fallen down. Thus the siliques/seeds were not separated but combined with the stems. At Viviez, a Zn-tolerant species S. vulgaris with its rhizospheric soils was sampled in triplicate to reveal any isotopic difference from N. caerulescens. The Zn isotopic signature of the local substrate was determined by collecting two parent rock samples. On the Vosges and Sainte Eulalie sites, the parent rock could not be sampled in the vicinity of the collected plants and soils. Sample Preparation and Analytical Methods. Soil and rock samples were dried in the laboratory at room temperature, ground, and put through a 200 μm sieve. Plants were separated into roots, stems, and leaves. Each plant organ was carefully rinsed with Milli-Q H2O (Millipore, 18.2 MΩ·cm), dried at 60 °C until a constant weight was attained, and then ground to a fine powder (500 μm sieve). The biomass of these plant organs was measured on a dry weight basis. The pH of rhizospheric soil was determined by use of 0.01 M CaCl2 extractant (soil:extractant = 1:5 m/v). 9973

dx.doi.org/10.1021/es3015056 | Environ. Sci. Technol. 2012, 46, 9972−9979


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Table 1. Zn Concentrations and Zn Isotopic Composition (δ66Zn) for Plant, Soil, and Rock Samplesa sample type and plant organ

plant biomass (g DW)b

plant 1, leaf plant 1, stem plant 1, root plant 2, leaf plant 2, stem plant 2, root plant 3, leaf plant 3, stem plant 3, root soil avail. soil Zn parent rock

0.77 1.29 1.47 1.23 2.5 6.83 0.91 1.19 2.36

leaf stem root soil avail. soil Zn parent rock

0.087 0.84 0.25

plant 1, leaf plant 1, stem plant 1, root plant 2, leaf plant 2, stem plant 2, root soil 1 soil 2 avail. soil Zn

0.47 0.29 0.073 0.23 0.4 0.034

leaf stem root soil avail. soil Zn

3.53 1.6 0.28

Zn concn (μg·g−1 DW)b Viviez site, S. vulgaris 772 452 954 864 516 801 490 410 1027 2220 7.26 212 Viviez site, N. caerulescens 8759 2505 2239 2396 11.53 185 Sainte Eulalie site, N. caerulescens 3922 1361 1184 12330 3135 1185 107 107 0.33 Vosges site, N. caerulescens 513 320 229 115 0.15

Fc 0.23 0.23 0.54 0.14 0.16 0.70 0.13 0.15 0.72

0.22 0.61 0.16

0.79 0.17 0.04 0.69 0.30 0.01

0.76 0.21 0.03

BCFd 0.35 0.20 0.43 0.39 0.23 0.36 0.22 0.18 0.46

3.66 1.05 0.93

36.7 12.7 11.1 115 29.3 11.1

4.45 2.78 1.98

δ66Zn (‰)

2σe

nf

0.11 0.09 0.01

0.48 0.39 0.44 0.47 0.38 0.47 0.48 0.28 0.39 0.48 0.51 0.36

0.03 0.08 0.05

2 2 3 1 1 1 1 1 1 4 3 3

0.17 0.39 0.62 0.22 0.44 0.45

0.12 0.09 0.06 0.02 0.11 0.03

3 2 2 3 2 5

0.03 0.16 0.43 −0.10 0.14 0.49 −0.01 0.07 0.16

0.07 0.08 0.11

0.11

2 2 2 1 1 1 2 1 4

0.07 0.09 0.04 0.09 0.08

2 2 4 3 2

0.59 0.65 0.70 −0.02 −0.13

0.01

a

Plant, soil, and rock samples were collected at Viviez (Zn-contaminated site), Sainte Eulalie (a granite noncontaminated site), and Vosges (a serpentine site). bDW, dry weight. cF is Zn fraction of plant organ, calculated as the ratio of Zn mass in plant organ (roots, stems, and leaves) to Zn mass in the whole plant. dBCF is bioconcentration factor, the ratio of Zn concentration in plant organ (roots, stems, and leaves) to that in soil. e2σ corresponds to the reproducibility of n isotopic measurements from the same solution. fn represents the number of isotopic measurements.

from the samples (>5 μg). Two international reference materials, BCR 62 (Olea europaea L.) and V464 (oak leaves), were used to ensure the accuracy and reproducibility of the procedure. For Zn isotope analysis, matrix components were separated from Zn on the AGMP1 (100−200 mesh, chloride form) anion-exchange resin (Bio-Rad Laboratories, Hercules, CA) as detailed in Cloquet et al.26 The purification procedure was repeated twice to completely remove Fe.27 After the removal of matrix and Fe by anion-exchange chemistry, the Zn fraction was dried, redissolved in 1 mL of 0.1 M HNO3, and placed in an ultrasonic bath for 30 min. Zinc isotopic measurements were carried out on a Neptune plus MC-ICP-MS (ThermoFinnigan) located at CRPG, Nancy (France). Instrumental mass bias was corrected according to the empirical method previously published,25 with Cu NIST 976 for Zn. In addition, the standard-sample-standard bracketing technique was applied along Zn isotopic measurements. Cu NIST 976 was added to the purified Zn fractions and a Cu (NIST 976) + Zn (IRMM-

To measure Zn concentration in soils and in plant tissues, samples were digested in concentrated HNO3−H2O2−HF− HCl mixtures on a hot plate at 100 °C for 72 h or until complete digestion. For analysis of available Zn in soils, ground soil samples (5 g) were shaken with 25 mL of 1 M CaCl2 for 24 h and then centrifuged at 2200 g for 20 min according to the method of Young et al.25 All digests and extracts were then evaporated to dryness, and the residues were dissolved in 1 mL of 6 M HCl. The resulting solutions were split into two parts: 0.1 mL for Zn concentration by ICP-MS (Perkin-Elmer ICPMS SCIEX Elan 6000 or Thermo X7) and 0.9 mL for isotope analysis. The water used for dilution and dissolution was obtained from a Millipore deionizing system at 18.2 MΩ·cm. Other reagents used for sample preparation (HCl and HNO3) were sub-boiled or bought directly as Suprapur (HF 48% v/v and H2O2). Along the digestion and purification processes, blank tests were performed to estimate the level of contamination induced by the treatment (always root. In terms of site, the BCFs of N. caerulescens followed the order of Sainte Eulalie > Vosges > Viviez. All the BCFs of N. caerulescens, particularly from Sainte Eulalie, were much higher than those of S. vulgaris, with BCFs well below 1. Zn Isotopes in Rock, Soil, and Plant Samples. Most rock, soil, and plant samples collected at Viviez, Sainte Eulalie,

Figure 1. Zn isotopic composition of the different plant parts (a) in Noccaea caerulescens and corresponding soil and rock collected at the Viviez, Sainte Eulalie, and Vosges sites and (b) in Silene vulgaris and corresponding soil and rock collected at the Viviez site. The error bars represent typical 2σ calculated from the average of 2σ (±0.08‰) for all the samples in Table 1. 9975



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Eulalie between root and stem (Δ66Znstem−root = −0.31‰), and in Viviez between stem and leaf (Δ66Znleaf−stem = −0.22‰) (Table S1, Supporting Information). By contrast, the fractionation was not isotopically different between the roots, stems, and leaves of S. vulgaris, which presented good consistency of isotopic compositions in the triple individuals. Average Zn Isotopic Composition in Plants. The average isotopic signature of Zn incorporated in the total plant was calculated according to the following equation: δ 66 Zn plant =

dissolution of goethite (α-FeOOH).33 In the present study, we speculate that this process may produce, if any, a small isotopic fractionation in terms of an insignificant or limited difference between the total soil and the CaCl2-extractable Zn (Table 1, Figure 1), which is supposed to be more plantrelevant. In process 2, some proportion of the Zn will adsorb on the root surface and become bound to cell walls. Adsorption of Zn may create negative or positive fractionation on inorganic surfaces34 and positive fractionation on organic surfaces.16,35,36 The fractionation appeared more pronounced in the marine diatoms with values of Δ66Znadsorption−media ranging from 0.08‰ to 0.45‰, which was supposed to link with structure of silica shells and stability of Zn complexes formed.35 However, the adsorption-generated enrichment in heavier Zn on root surface would be smaller in comparison with diatoms based on the following observations: (i) In rice, tomato, and lettuce grown in a nutrient solution, there was no change in the isotopic composition of roots (Δ66Znroot−solution = 0.08−0.18‰) leached with LaCl3, which is used to remove apoplastically bound Zn.16 (ii) Recently Aucour et al.21 suggested that Zn isotope compositions in the roots of the Zn hyperaccumulator Arabidopsis halleri and its nonaccumulating relative A. petraea should not show any great difference, if adsorption on the root surfaces played an important role in heavy Zn enrichment in roots. Therefore, the change of Zn isotopic composition from soil to root in N. caerulescens is presumably determined by process 3, in which Zn uptake into root symplast is mediated by a combination of a high-affinity (e.g., ZIP family protein) and/or a low-affinity (e.g., ion-channel and/or electrogenic pumps) transport system. High-affinity transport should favor the heavy isotopes, while the reverse is true for low-affinity transport.16 High- or low-affinity transport depends on the concentration of free Zn ions in the external media and will ultimately determine the isotopic signatures of bulk Zn taken up into the whole plants. In wheat, Hacisalihoglu et al.37 suggested that Zn solutions with a concentration lower than 10 nM should trigger high-affinity transport, while above this threshold low-affinity transport will be dominant. In N. caerulescens, though physiological and molecular studies have shown a high-affinity transporter (ZNT1) mediated Zn uptake with a Michaelis− Menten constant (Km) of 6−8 μM,8,12 the threshold for the switch between high- and low-affinity transport has not been characterized. In our experiment (Table 1), the strong fractionation toward heavy Zn between plant and soil in Vosges (Δ66Znplant−soil = 0.63‰) therefore suggests that Zn uptake is predominantly mediated by high-affinity transporter proteins, that is, ZNT1. This is consistent with the molecular study12 demonstrating much higher expression of ZNT1 in the roots of N. caerulescens exposed to Zn deficiency (0−1 μM Zn) than those exposed to Zn excess. By comparison, the individuals from Viviez and Saint-Eulalie sites showed slightly heavy (0.16‰) and neutral (−0.01‰) fractionation relative to soil, respectively, indicating that Zn uptake is probably via both high- and low-affinity transport mechanisms, yet that the overall isotopic compositions are counterbalanced. Moreover, the higher Δ66Znplant−soil from Vosges, compared to Viviez and Sainte Eulalie, suggests that the isotopic variation is probably controlled by the available Zn supplied from the soil. In fact, the lowest available Zn (0.15 μg·g−1, Table 1) was found in the Vosges soil, where the ZIP transporters on the root membrane might be activated

∑i miciδ 66Zni ∑ mici

where mi and ci are the mass and Zn concentration of plant part i (root, stem, or leaf). The average δ66Znplant of N. caerulescens was heavier than that of rhizospheric soil except in Sainte Eulalie, where the average isotopic composition of the plant was similar to that of the soil (Table S1, Supporting Information). In Vosges, N. caerulescens showed the highest enrichment with Δ66Znplant−soil = +0.63‰. On average, S. vulgaris exhibited no fractionation of Zn isotopes relative to soil.

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DISCUSSION Fractionation during Zn Uptake from Soil to Plant of N. caerulescens. In N. caerulescens, Zn isotopic mass balance yields average isotopic fractionation between plant and soil Zn with Δ66Znplant−soil values ranging from −0.01‰ to 0.63‰ at the three sites (Table S1, Supporting Information), although they showed a similar fractionation pattern with an enrichment in heavy isotopes of 0.40−0.72‰ from soil to root, followed by a depletion from root to shoot (will be discussed in the next section). Isotopic fractionation during Zn uptake depends essentially on the interaction within soil−root interface, probably controlled by at least three processes: mineral mobilization by root exudates (process 1), adsorption of Zn on root apoplast (e.g., through cell wall binding) (process 2), and Zn transport across the plasma membrane into the root symplast (process 3). In this study, it is hard to distinguish between the isotopic fractionation of Zn at these processes, but it was possible to constrain the isotope effect occurring in each process. In process 1, a possibility for fractionation is related to the excretion of root exudates for Zn mobilization in soil and thus for efficient Zn uptake. The substantially increased release of phytosiderophores (PS) has been reported in roots of wheat30 and other wild grass species.31 In the field-grown rice, Arnold et al.14 observed a heavy Zn enrichment in roots and shoots relative to plant-available Zn. They suggested with a mathematical model that the secretion of PS deoxymugineic acid for Zn solubilization in soil and further uptake of PS-Zn by root, which tend to produce heavy Zn discrimination, is the only plausible explanation. Nevertheless, it is very unlikely that N. caerulescens also adopts this strategy because PS secretion is observed only in monocotyledon (e.g., graminaceous) species, especially in Zn-deficient soil. Moreover, Zhao et al.32 found that root exudates from Ganges and Prayon ecotypes of N. caerulescens do not significantly enhance Zn mobilization either in Zn-loaded resin or in contaminated calcareous soil. Although a significant role of PS is not expected in the field-grown N. caerulescens, we cannot rule out the possible change of isotopic compositions originating from mineral dissolution, as shown in a ligand-controlled fractionation for Fe isotopes during 9976



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by extremely low Zn supply.12 It is noticeable that since available Zn in the soil depends strongly on soil properties, that is, soil pH, we observed a linear correlation between Δ66Znplant soil/available soil and soil pH (P < 0.001; Figure S2, Supporting Information), suggesting that soil pH may play a role in the observed isotopic difference among individuals from the three sites. Fractionation during Zn Translocation in N. caerulescens. The more negative values of δ66Zn in stems and leaves of N. caerulescens in relation to that of roots (Figure 1; Table S1, Supporting Information) is consistent with previous studies suggesting that Zn translocation from root to shoot favors the light Zn isotopes.17−20 Long-distance translocation of Zn in N. caerulescens requires first the active loading of Zn into the xylem and is presumed to depend on upregulation of a heavy metal ATPases (HMAs).13,38 NcHMA4 is a member of the P-type ATPases that transport Zn against the electrochemical gradient by using the energy supplied by ATP hydrolysis,38 which should favor the light isotopes because of its greater diffusion coefficient.16 In N. caerulescens, NcHMA4 is primarily expressed to a greater extent in roots than is the case for Zn-sensitive relative Arabidopsis thaliana. This strongly supports the hypothesis that HMA4 plays an important role in Zn translocation and hyperaccumulation.13 The next step regarding Zn upward movement is associated with Zn chelation with organic ligands in xylem sap.38,39 Though it is still unclear which specific ligands are involved, our results support a previous study based on X-ray absorption spectroscopy, which tend to show that most Zn in the xylem sap is present as free hydrated Zn2+ ions, while a small part is bound to organic acids.40 A negative correlation is proposed between the length of the plants and the extent of Zn fractionation,17 with the fractionation per distance of −0.005‰ and −0.012‰ cm−1 in P. australis under Zn-sufficient and Zn-toxic conditions, respectively,19 and −0.006 ‰ cm−1 in bamboo.18 We are not able to examine this correlation with the present data, but as a rosette plant with an average height of 20 cm, N. caerulescens appears to have wide variation with much higher Δ66Znleaf−root per distance in the individuals of Viviez (−0.022‰ cm−1) and Sainte Eulalie (−0.025‰ cm−1 ) than that of Vosges (−0.005‰ cm −1 ), P. australis,13 and bamboo. 12 This observation may be reflected by the ability of Zn accumulation in plant organs of N. caerulescens, the higher Zn concentration in the leaves, and the more negative δ66Zn value obtained (Figure S3, Supporting Information). Therefore, the extent of Zn fractionation in shoots can be species-specific and modified both by the ability of Zn translocation and by the morphological characteristics of plant species (e.g., plant height and/or life form). In N. caerulescens, the roots were enriched in heavy isotopes relative to whole plant Zn with Δ66Znroot−plant values ranging from 0.09‰ to 0.52‰ (Table S1, Supporting Information). This allowed us to estimate the Zn fractionation in N. caerulescens and S. vulgaris using a Rayleigh-type mass balance, where the isotopic composition of root Zn pool depends on the Zn initially taken up from soil (≈δ66Znplant) and the Zn proportion left in root after translocation (Froot): Δ66Znroot−plant = δ 66 Zn r o o t − δ 6 6 Zn p l a n t = Δ 6 6 Zn t r a n s l o c a t i o n ln F r o o t (Δ66Zntranslocation represents the isotope discrimination during translocation process in the shoots). The data produced a good fit between Δ66Znroot−plant and ln Froot (Figure 2), thereby suggesting that a large proportion of Zn enrichment in roots was due to the translocation process. Unexpectedly, we

Figure 2. A Rayleigh mass balance to model the Zn isotopic fractionation between root and whole plant (Δ66Znroot−plant), as a function of Froot during translocation. Froot is given as the ratio of Zn mass in the root to Zn mass in whole plant: Froot = [Zn]root/[Zn]plant. The error bars represent typical 2σ (±0.08‰) as defined in Figure 1a.

observed a relatively small fractionation between root and plant (Δ66Znroot−plant = 0.09‰) in the individual at Vosges site, which should have a Δ66Znroot−plant of 0.31‰ according to the model. It is important to consider that a Rayleigh model approach always assumes that the substrate is a homogeneous well-mixed pool in which all parts are equally affected by the removal of fractionated material. However, there were probably different Zn pools with varying mobilities in different organs of the lower plant parts. If a certain (more mobile) Zn pool is quantitatively translocated, no net fractionation can be expected. Thus, the finding that some samples did not agree with the Rayleigh model could be explained, for instance, by a different relative extent of translocation of different Zn pools within the plants. Mechanisms Involved in N. caerulescens Compared to S. vulgaris. S. vulgaris is a heavy-metal-tolerant grass originating from different metallicolous sites in Europe.41 Unlike normal plants, in which metal tolerance is mainly achieved by a restriction of metal uptake, S. vulgaris collected from the Viviez site allowed high Zn accumulation in roots but further transferred a limited amount of Zn in the above-ground parts (Table 1). This characteristic, contrary to the behavior of N. caerulescens, requires an efficient sequestration mechanism for excess Zn in the roots, for example, localization in cell walls, intercellular spaces, and vacuoles.18 Correspondingly, S. vulgaris indeed exhibited a distinct pattern of isotopic fractionation compared with that of N. caerulescens predominantly in two aspects (Figure S4, Supporting Information). First, there was no significant difference in δ66Zn between the roots of S. vulgaris and its rhizospheric soils, which differs largely from the positive values of Δ66Znroot−medium so far reported in higher plants.14,16−19 Moreover, the difference in δ66Znplant soil between S. vulgaris and N. caerulescens was −0.21‰, implying that Zn uptake by roots of S. vulgaris is probably through a transport system with no isotope selection under Zn excess conditions; the expression of transporter proteins (e.g., ZNT1) in this species might be depressed compared to N. caerulescens. Second, the transfer of Zn from root to stem and stem to leaf in S. vulgaris also produced no significant isotope fractionation. This observation was not expected, since the root sequestration processes, like adsorption on root cell walls and complexation in vacuoles should favor heavy enrichment of isotopes, resulting in a lighter Zn translocation to the aboveground parts. Therefore, this indicates that the translocation in 9977



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S. vulgaris is a quantitative process; it is likely that the translocation processes (e.g., diffusion and/or complexation in xylem sap) would not induce any fractionation or these processes offset together. Therefore the shoots would conserve the isotopic composition of the roots. Environmental Implications. Several implications can be inferred from the present study. First, the isotopic fractionation during Zn uptake created large differences between the individuals of N. caerulescens from three sites, with Δ66Znplant−soil values ranging from −0.01‰ to 0.63‰, although they showed a similar fractionation pattern from soil to root and root to shoot. The bulk Zn isotopic compositions in plants could therefore be used to distinguish Zn uptake strategies, which are probably locality-specific. Furthermore, the intensity of fractionation is likely to be mediated by soil conditions such as soil pH, total Zn content, and/or bioavailable Zn, indicating a potential utilization of isotopic signatures in Zn nutritional studies. More data from field plants are needed to test this hypothesis. Second, Zn transfer from roots toward xylem is an active unidirectional process, which allowed us to estimate the isotopic fractionation during Zn translocation using a Rayleightype mass balance. Plotting Δ66Znroot/shoot−plant as a function of the Zn mass fraction in shoots (Fshoot) with our present results and previous published data produced two exponential curves with a good fit between Δ66Znroot−plant and Fshoot for two Arabidopsis species,21 N. caerulescens and S. vulgaris from our present study. The other plant species (rice, tomato, lettuce, and wheat)16,20 are within the curves (Figure 3). This

to-shoot transfer was about 0.40‰. Once more studies further quantify this fractionation factor precisely, Zn isotope signatures can be used to estimate the allocation of Zn pool in the underground and aerial part of plant. Third, it is worth noticing that S. vulgaris, with Δ66Znshoot−plant values around 0, did not fit this general model, indicating that this species appears to have a particular Zn homeostasis. This observation therefore warrants more investigations by a combination of stable isotopes and physiological techniques on the biomolecular mechanisms responsible for root absorption, sequestration, and translocation. A similar observation can be made with N. caerulescens from the serpentine site Vosges, which might also have specific mechanisms regarding Zn accumulation.

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ASSOCIATED CONTENT

S Supporting Information *

One table, listing Zn isotopic fractionation and average Zn isotopic composition, and four figures, showing δ68Zn vs δ66Zn, correlation between rhizospheric soil pH and isotopic composition, correlation of Zn isotopic composition with Zn concentration in plant organs and their corresponding BCFs, and comparison of Zn isotopic composition in plant organs, rhizospheric soils, and rocks. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Catherine Sirguey and Cédric Gonneau for their assistance in sample collecting, and Aimeryc Schumacher for his help with MC-ICP-MS measurements. We also thank the Institut National de la Recherche Agronomique (INRA) for its support, as well as the Observatoire des Sciences de l’Univers (OSU OTELo) of Université de Lorraine for funding the analysis. Y.-T.T. gratefully acknowledges the grant by China Scholarship Council (CSC) and the support by Natural Science Foundation of China (40901151, U0833004). We are grateful to the three anonymous reviewers for their valuable comments and suggestions.

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REFERENCES

(1) Broadley, M. R.; White, P. J.; Hammond, J. P.; Zelko, I.; Lux, A. Zinc in plants. New Phytol. 2007, 173 (4), 677−702. (2) Reeves, R. D.; Brooks, R. R. European species of Thlaspi L. (Cruciferae) as indicators of nickel and zinc. J. Geochem. Explor. 1983, 18 (3), 275−283. (3) Chaney, R. L.; Malik, M.; Li, Y. M.; Brown, S. L.; Brewer, E. P.; Angle, J. S.; Baker, A. J. M. Phytoremediation of soil metals. Curr. Opin. Biotechnol. 1997, 8 (3), 279−284. (4) Salt, D. E.; Smith, R. D.; Raskin, I. Phytoremediation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998, 49 (1), 643−668. (5) Pilon-Smits, E. Phytoremediation. Annu. Rev. Plant Biol. 2005, 56 (1), 15−39. (6) Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. Zinc and cadmium uptake by hyperaccumulator Thlaspi caerulescens and metal tolerant Silene vulgaris grown on sludge-amended soils. Environ. Sci. Technol. 1995, 29 (6), 1581−1585. (7) Shen, Z. G.; Zhao, F. J.; McGrath, S. P. Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-

Figure 3. Zn isotopic fractionation between root/shoot and whole plant (Δ66Znroot/shoot−plant) as a function of Fshoot in higher plants. Fshoot is given as the ratio of Zn mass in the shoot to Zn mass in the whole plant: Fshoot = [Zn]shoot/[Zn]plant. Fshoot = 1 − Froot. Data were adapted from the present study and refs 16, 20, and 21.

observation suggests that isotopic fractionation during translocation creates in roots a heavy Zn enrichment that can vary according to conditions and plant species tested. We further tested a series of fractionation factors (ε ≈ δ66Znshoot − δ66Znroot) and found a ε value of −0.4‰ better fit the overall observed fractionation in these plant species, and hence we estimated that the average fractionation factor during the root9978



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hyperaccumulator Thlaspi ochroleucum. Plant Cell Environ. 1997, 20 (7), 898−906. (8) Lasat, M. M.; Baker, A.; Kochian, L. V. Physiological characterization of root Zn2+ absorption and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiol. 1996, 112 (4), 1715−1722. (9) Lasat, M. M.; Baker, A. J. M.; Kochian, L. V. Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens. Plant Physiol. 1998, 118 (3), 875−883. (10) Kü p per, H.; Jie Zhao, F.; McGrath, S. P. Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiol. 1999, 119 (1), 305−312. (11) Frey, B.; Keller, C.; Zierold, K. Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens. Plant Cell Environ. 2000, 23 (7), 675−687. (12) Pence, N. S.; Larsen, P. B.; Ebbs, S. D.; Letham, D. L. D.; Lasat, M. M.; Garvin, D. F.; Eide, D.; Kochian, L. V. The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (9), 4956− 4960. (13) Papoyan, A.; Kochian, L. V. Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance: characterization of a novel heavy metal transporting ATPase. Plant Physiol. 2004, 136, 3814−823. (14) Arnold, T. I. M.; Kirk, G. J. D.; Wissuwa, M.; Frei, M.; Zhao, F.J.; Mason, T. F. D.; Weiss, D. J. Evidence for the mechanisms of zinc uptake by rice using isotope fractionation. Plant Cell Environ. 2010, 33 (3), 370−381. (15) von Blanckenburg, F.; von Wiren, N.; Guelke, M.; Weiss, D. J.; Bullen, T. D. Fractionation of metal stable isotopes by higher plants. Elements 2009, 5 (6), 375−380. (16) Weiss, D. J.; Mason, T. F. D.; Zhao, F. J.; Kirk, G. J. D.; Coles, B. J.; Horstwood, M. S. A. Isotopic discrimination of zinc in higher plants. New Phytol. 2005, 165 (3), 703−710. (17) Viers, J.; Oliva, P.; Nonell, A.; Gélabert, A.; Sonke, J. E.; Freydier, R.; Gainville, R.; Dupré, B. Evidence of Zn isotopic fractionation in a soil-plant system of a pristine tropical watershed (Nsimi, Cameroon). Chem. Geol. 2007, 239 (1−2), 124−137. (18) Moynier, F.; Pichat, S.; Pons, M.-L.; Fike, D.; Balter, V.; Albarède, F. Isotopic fractionation and transport mechanisms of Zn in plants. Chem. Geol. 2009, 267 (3−4), 125−130. (19) Caldelas, C.; Dong, S.; Araus, J. L.; Jakob Weiss, D. Zinc isotopic fractionation in Phragmites australis in response to toxic levels of zinc. J. Exp. Bot. 2011, 62 (6), 2169−2178. (20) Jouvin, D.; Weiss, D. J.; Mason, T. F. M.; Bravin, M.; Louvat, P.; Zhao, F. J.; Ferec, F.; Hinsinger, P.; Benedetti, M. F. Stable isotopes of Cu and Zn in higher plants: evidence for Cu reduction at the root surface and two conceptual models for isotope fractionation processes. Environ. Sci. Technol. 2012, 46, 2652−2660. (21) Aucour, A. M.; Pichat, S.; Macnair, M. R.; Oger, P. Fractionation of stable zinc isotopes in the zinc hyperaccumulator Arabidopsis halleri and nonaccumulator Arabidopsis petraea. Environ. Sci. Technol. 2011, 45 (21), 9212−9217. (22) Reeves, R. D.; Schwartz, C.; Morel, J. L.; Edmondson, J. Distribution and metal-accumulating behavior of Thlaspi caerulescens and associated metallophytes in France. Int. J. Phytoremed. 2001, 3 (2), 145−172. (23) Chardot, V.; Echevarria, G.; Gury, M.; Massoura, S.; Morel, J. L. Nickel bioavailability in an ultramafic toposequence in the Vosges Mountains (France). Plant Soil 2007, 293, 7−21. (24) Dinesh, R.; Srinivasan, V.; Hamza, S.; Parthasarathy, V. A.; Aipe, K. C. Physico-chemical, biochemical and microbial peroperties of the rhizospheric soils of tree species used as supports for black pepper cultivation in the humid tropics. Geoderma 2010, 158, 252−258. (25) Young, S. D.; Tye, A.; Carstensen, A.; Resende, L.; Crout, N. Methods for determining labile cadmium and zinc in soil. Eur. J. Soil Sci. 2000, 51, 129−136.

(26) Cloquet, C.; Carignan, J.; Libourel, G. Isotopic composition of Zn and Pb atmospheric depositions in an urban/periurban area of northeastern France. Environ. Sci. Technol. 2006, 40 (21), 6594−6600. (27) Cloquet, C.; Carignan, J.; Lehmann, M.; Vanhaecke, F. Variation in the isotopic composition of zinc in the natural environment and the use of zinc isotopes in biogeosciences: a review. Anal Bioanal. Chem. 2008, 390 (2), 451−463. (28) Meerts, P.; Duchêne, P.; Gruber, W; Lefèbvre, C. Metal accumulation and competitive ability in metallicolous and nonmetallicolous Thlaspi caerulescens fed with different Zn salts. Plant Soil 2003, 249, 1−8. (29) Sivry, Y.; Riotte, J.; Sonke, J. E.; Audry, S.; Schäfer, J.; Viers, J.; Blanc, G.; Freydier, R.; Dupré, B. Zn isotopes as tracers of anthropogenic pollution from Zn-ore smelters in the Riou Mort− Lot River system. Chem. Geol. 2008, 255, 295−304. (30) Cakmak, I.; Sari, N.; Marschner, H.; Ekiz, H.; Kalayci, M.; Yilmaz, A.; Braun, H. J. Phytosiderophore release in bread and durum wheat genotypes differing in zinc efficiency. Plant Soil 1996, 180, 183− 189. (31) Carmak, I.; Ö ztürk, L.; Karanlik, S.; Marschner, H.; Ekiz, H. Zinc-efficient grasses enhance release of phytosiderophores under zinc deficiency. J. Plant Nutr. 1996, 19, 551−563. (32) Zhao, F. J.; Hamon, R. E.; McLaughlin, M. J. Root exudates of the hyperaccumulator Thlaspi caerulescens do not enhance metal mobilization. New Phytol. 2001, 151 (3), 613−620. (33) Wiederhold, J. G.; Kraemer, S. M.; Teutsch, N.; Borer, P. M.; Halliday, A.; Kretzschmar, R. Iron isotope fractionation during protonpromoted, ligand-controlled, and reductive dissolution of geothite. Environ. Sci. Technol. 2006, 40, 3787−3793. (34) Pokrovsky, O. S.; Viers, J.; Freydier, R. Zinc stable isotope fractionation during its adsorption on oxides and hydroxides. J. Colloid Interface Sci. 2005, 291, 192−200. (35) Gélabert, A.; Pokrovsky, O. S; Viers, J.; Schott, J.; Boudou, A.; Feurtet-Mazel, A. Interaction between zinc and freshwater and marine diatom species: Surface complexation and Zn isotope fractionation. Geochim. Cosmochim. Acta 2006, 70, 839−857. (36) John, S. G.; Geis, R. W.; Saito, M. A.; Boyle, E. A. Zinc isotope fractionation during high-affinity and low-affinity zinc transport by the marine diatom Thalassiosira oceanica. Limnol. Oceanogr. 2007, 52, 2710−2714. (37) Hacisalihoglu, G.; Hart, J. J.; Kochian, L. V. High- and lowaffinity zinc transport systems and their possible role in zinc efficiency in bread wheat. Plant Physiol. 2001, 125, 456−463. (38) Verbruggen, N.; Hermans, C.; Schat, H. Molecular mechanisms of metal hyperaccumulation in plants. New Phytol. 2009, 181 (4), 759−776. (39) Milner, M. J.; Kochian, L. V. Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system. Ann. Bot. 2008, 102 (1), 3−13. (40) Salt, D. E.; Prince, R. C.; Baker, A. J. M.; Raskin, I.; Pickering, I. J. Zinc ligands in the metal hyperaccumulator Thlaspi caerulescens as determined using X-ray absorption spectroscopy. Environ. Sci. Technol. 1999, 33 (5), 713−717. (41) Schat, H.; Vooijs, R.; Kuiper, E. Identical major gene loci for heavy metal tolerances that have independentaly evolved in different local populations and subspecies of Silene vulgaris. Evolution 1996, 50 (5), 1888−1895.

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Fractionation of Stable Zinc Isotopes in the Field-Grown Zinc

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