Environ. Sci. Technol. 2010, 44, 2904–2910
Uptake, Localization, and Speciation of Cobalt in Triticum aestivum L. (Wheat) and Lycopersicon esculentum M. (Tomato) RICHARD N. COLLINS,† ` RE,‡ ESTELLE BAKKAUS,‡ MARIE CARRIE HICHAM KHODJA,‡ OLIVIER PROUX,§ JEAN-LOUIS MOREL,| AND B A R B A R A G O U G E T * ,‡,⊥ UNSW Water Research Centre, School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia, Laboratoire Pierre Su ¨ e, CEA-CNRS UMR 9956, CEA Saclay, 91191 Gif-sur-Yvette, France, Observatoire des Sciences de l’Univers de Grenoble, ` UMS CNRS, 38400, St Martin d’Heres, France, Laboratoire Sols et Environnement, INPL(ENSAIA)-INRA UMR 1120, and BP 172, 2 avenue de la foreˆt de Haye, 54505 ` Vandoeuvre-les-Nancy Cedex, France
Received May 25, 2007. Revised manuscript received March 7, 2010. Accepted March 10, 2010.
The root-to-shoot transfer, localization, and chemical speciation of Co were investigated in a monocotyledon (Triticum aestivum L., wheat) and a dicotyledon (Lycopersicon esculentum M., tomato) plant species grown in nutrient solution at low (5 µM) and high (20 µM) Co(II) concentrations. Cobalt was measured in the roots and shoots by inductively coupled plasma-mass spectrometry. X-ray absorption spectroscopy measurements were used to identify the chemical structure of Co within the plants and Co distribution in the leaves was determined by microPIXE (particle induced X-ray emission). Although the root-toshoot transport was higher for tomato plants exposed to excess Co, both plants appeared as excluders. The oxidation state of Co(II) was not transformed by either plant in the roots or shoots and Co appeared to be present as Co(II) in a complex with carboxylate containing organic acids. Cobalt was also essentially located in the vascular system of both plant species indicating that neither responded to Co toxicity via sequestration in epidermal or trichome tissues as has been observed for other metals in metal hyperaccumulating plants.
Introduction Cobalt is not classified as an essential element for higher plants, however, it has been described as beneficial (1). At high concentrations Co is toxic for most vegetal species. Although previous studies have reported the symptoms of Co phytotoxicity, as well as toxicity thresholds, they have primarily been limited to dicotyledonous plants (2-4). * Corresponding autho phone: +33 (0)1 4977 1331; fax: +33 (0)1 4977 1354; email:
[email protected]. † University of New South Wales. ‡ Laboratoire Pierre Su ¨ e. § Observatoire des Sciences de l’Univers de Grenoble. | Laboratoire Sols et Environnement. ⊥ Present Address: AFSSA, Scientific Department, 27-31 Avenue du Ge´ne´ral Leclerc, 94701 Maison Alfort, France. 2904
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Plants generally possess regulation mechanisms to minimize the damage from exposure to nonessential metals. The uptake, distribution, and detoxification of metal ions are provided by, respectively, metal transport, chelation, and sequestration activities (5). However, only some of these mechanisms have been identified and only for a relatively few metals. X-ray absorption spectroscopy (XAS) has been used to determine the chemical speciation of several metals in plants and has resulted in the identification of certain detoxification mechanisms, for example, metal-induced production of phytochelatins or metal complexation by organic acids (6, 7). XAS consists of two complementary techniques: X-ray absorption near edge structure (XANES) which provides information such as the oxidation state of the probed element and extended X-ray absorption fine structure (EXAFS) which gives information on its coordination environment, such as the nature and the distance of the nearest neighboring atoms. Information on the speciation of metals in nonhyperaccumulator plants is lacking (8) and XAS studies on the chemical structure of Co in higher plants have not been reported. Distribution patterns of 60Co in plants were first investigated using autoradiographic techniques (9, 10). More recently, micro-PIXE has also been used for constructing two-dimensional maps of elemental localization in plants (11, 12). This technique has been used to show that some metals, like Ni, Cd, and Zn, are sequestered in leaf trichomes (13, 14) or in the leaf epidermis (15) of hyperaccumulator species. Studies concerning the distribution of Co in higher plants using PIXE technique are scarce (16, 17). In this study, the response of higher plants from the two classes of angiosperms: wheat (a monocotyledon) and tomato (a dicotyledonous species); to Co exposure was investigated. The uptake, chemical structure, and leaf localization of Co were examined with plants grown in nutrient solution at two Co levels: low and high dose exposure. This information is essential for comprehending the biological response of higher plants to Co stress.
Materials and Methods Plant Cultures. Seeds of Lycopersicon esculentum M. (tomato) and Triticum aestivum L. (winter wheat) were germinated for 7 days in a mixture of moistened vermiculite and sand. Seedling roots were briefly washed with nutrient solution and then transferred to the same nutrient solution composed of 2 mM K2SO4 and Ca(NO3)2, 1 mM MgSO4, 0.5 mM KH2PO4, 10 µM H3BO3, 5 µM MnSO4, 0.5 µM ZnSO4, 0.2 µM Na2MoO4, 0.5 µM CuSO4, and 20 µM Fe(III)EDTA. The pH was adjusted to 6.5 with NaOH and the solutions were changed every 2 days. Plants were precultured in this solution for 5 days and subsequently grown in nutrient solutions spiked with cobalt nitrate at 0, 5, and 20 µM Co concentrations (Sigma-Aldrich, Saint-Quentin-Fallavier, France). Each treatment was applied to six plants and experiments were replicated four times. All plants were grown in a growth chamber for a further 20 days with the following climatic conditions: day/night photoperiod, 16/8 h; light intensity, 310 µmol.m-2.s-1 (fluorescent tubes); temperature (day/night), 24/20 ( 1 °C and; relative humidity (day/night), 70/75%. Total Cobalt Concentrations in the Plants. Plant shoots were washed three times with ultrapure water and ground in liquid nitrogen with an agate mortar and pestle. The roots of the plants were immersed in a 0.01 M-Na2EDTA solution for 30 s to remove adsorbed metals, before being rinsed and ground in an identical fashion to the shoots. Plant samples 10.1021/es903485h
2010 American Chemical Society
Published on Web 03/26/2010
were then oven-dried at 60 °C for 48 h and weighed. Triplicate samples of 50-100 mg of the plant materials (5 mg for the roots of tomato plants from the 50 µM Co-treatment) were acid digested in a microwave system (Microdigest A301; Prolabo, Fontenay-sous-Bois, France) using Normatom quality grade reagents: HNO3, H2O2, HF (VWR International, Fontenay-sous-Bois, France). Blank samples and a reference standard (Lichen-336, IAEA, Vienna, Austria) were prepared and analyzed using the same conditions. Cobalt concentrations were determined by quantifying 59Co concentrations using an X7 Series quadrupole ICP-MS instrument (Thermo Electron Corporation, Cergy-Pontoise, France), using collision cell technology and 9Be and 103Rh as internal standards (1 µg · L-1). Determination of the Coordination Environment of Co by XAS. Freeze-dried samples and Co references (Co(III)acetylacetonate CoIII(C5H7O2)3 for Co(III)-O; Co(II)acetate CoII(C2H3O2)2 for Co(II)-O; and Co(II)Nitrate CoII(NO3)2 · 6H2O for Co(II)-N/O), were all pressed into 5-mm diameter pellets for XAS analyses. Fresh plant leaf samples were also analyzed to identify any shifts in Co speciation that may possibly arise from freeze-drying. Experiments were performed at the Co K-edge (7.709 keV) on the BM30b beamline of the european synchrotron radiation facility, Grenoble, France (18). The storage ring was operated in hybrid mode (24 × 8 bunches) at 6 GeV with a ∼200 mA current. The beam energy was selected using a Si(220) double-crystal monochromator with an experimental resolution close to that theoretically predicted (∼0.4 eV) (19). The beam size on the sample was approximately 300 × 200 µm (H × V). Spectra were recorded in transmission mode for the Co reference materials while data for the plant materials were collected in fluorescence mode using a 30-element solid state Ge detector (Canberra, St Quentin Yvelines, France). XANES spectra were normalized and EXAFS curves extracted using the Athena code (20). The resulting EXAFS curves in the wavevector (k) space were weighted by k2, Fourier-transformed with a data range from k ) 3 to 13.7 Å-1 and converted into pseudointeratomic distance space (R). Fourier transformation facilitates filtering of the first shell contributions (i.e., the closest neighboring atoms surrounding Co). The k2-weighted EXAFS spectra were fitted using the Artemis code based on the IFEFFIT program (21). Fits of the filtered spectra were performed in k space using a priori CosO, CosN, and CosS theoretical functions. Refinement of the spectra was completed by least-squares minimization and the EXAFS structural parameters for each shell (chemical nature of the neighboring atoms, coordination number, interatomic distance and Debye-Waller factor) were estimated. Determination of Spatial Distribution of the Elements by Micro-PIXE. Five to 10 samples were randomly selected from the old leaves of wheat and tomato plants. Fresh leaf samples were rinsed in ultrapure water before being cut and mounted onto carbon tape. The samples were then cryofixed in isopentane chilled to -160 °C and freeze-dried at -10 °C for 24 h under a vacuum of 0.37 mbar. These dehydrated samples were stored in a desiccator until micro-PIXE analyses. The nuclear microprobe experiments were performed with a 3 MeV proton beam at the Pierre Su ¨ e Laboratory facility (22). The beam was focused to 3 × 3 µm2 with an average intensity of 500 pA. The PIXE spectra were collected using a Si(Li) detector positioned at an angle of 45° from the target. The active surface was restricted by a circular collimator of 6 mm diameter. A 250 µm Mylar film was positioned in front of the detector to attenuate X-rays from the light elements. The distance sample-detector was optimized to maintain PIXE count rates below 1000 counts · s-1. Total deposited charges ranged from 1 to 7 µC. Raw elemental distribution maps were completed by extracting total counts for the corresponding X-ray energy
windows using the RISMIN software (23). Off-line image processing was then performed to extract spectra from selected regions. Elemental quantification from the PIXE spectra was achieved using GUPIX (24), either from the spectrum of the total scan or from spectra extracted from regions of interest in the scan. For these analyses the composition of the sample matrices was assumed to be identical to the generic plant tissue composition: H-6% C-45% O-45%, N-1.5% K-1% Ca-0.5% d.w. (chemical formula: H6000C4000O3000N100K25Ca12) (25). This method assumes infinite sample thickness for the analysis of whole leaves.
Results Cobalt Phytotoxicity. Visible symptoms of toxicity for the 5 µM-Co treatment were limited to a slight inhibition of shoot greening which is similar to the visual observations reported by Bakkaus et al. (16). The shoots of the plants exposed to 20 µM-Co were clearly yellowed for both species. The young leaves of the plants developed chlorosis in the interveinal areas, appearing initially in the basal part of the expanding leaves, and later, the emerging leaves showed distinct interveinal chlorosis of the whole lamina. In addition, necrotic spots were observed on the chlorotic portions of the tomato plants. Although symptoms of toxicity were not visually apparent at 5 µM Co, biomass tolerance index (TI) calculations, the ratio between the dry weight biomass for a Co treatment and the respective biomass of the control, indicated that this concentration of Co was sufficient to decrease the biomass production of both plant species (Supporting Information (SI) Figure S1). Whereas wheat TIs were similar at 5 and 20 µM Co concentrations, the tomato TI was drastically reduced by increasing the nutrient solution Co concentration to 20 µM. The results obtained here with wheat are in accordance with previous experiments (16), however, the biomass production for the tomato plants is significantly lower. We attribute this dissimilarity to the use of different tomato varieties used here (Grosse Lisse) and those in the experiments reported by Bakkaus et al. (16) (Roma VF). Cobalt Uptake and Root-to-Shoot Transport. Total Co concentrations in the shoots and roots of the plants are reported in SI Table S1. For the two plants the concentration of Co increased in both the shoots and roots with increasing Co supply in the nutrient solution. In addition, Co accumulation was higher in the roots than in the shoots. Cobalt concentrations in the wheat and tomato shoots were of the same order of magnitude. The concentration of Co in the wheat roots was enhanced by a factor of 2.4 between the 5-20 µM-Co treatments and by 1.3 for the tomato roots. In contrast, the ratio of Co concentrations (shoot:root) between the 5-20 µM-Co treatments was constant for wheat whereas it increased by a factor 2 for tomato. In order to account for biomass production, the total Co concentrations were normalized to total shoot and root dry weights (SI Table S1). For wheat, the Co content of the roots represented 88 and 90% of the total Co plant content for, respectively, the 5 and 20 µM Co treatments, whereas it was 89 and 48% for the tomato plants. Therefore, Co was preferentially located in the roots of both plants at 5 µM Co concentrations. Although Co was also retained in the roots of wheat at the 20 µM Co treatment, it was translocated from the roots to shoots of tomato resulting in similar Co contents throughout the plant. Cobalt Speciation Transformations during Internal Translocation. X-ray absorption spectroscopy spectra of plant samples after exposure to Co(II) are presented in Figure 1. The spectra for the freeze-dried samples were not significantly different from the fresh samples (data not shown), although the signal-to-noise ratio was higher for the fresh samples. The normalized XANES spectra for the freezedried plant samples and reference compounds are presented VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. (a) Co K-edge normalized XANES spectra of tomato and wheat plants exposed to 20 µM Co and of Co reference compounds (Co(III)Acetylacetonate for Co(III), Co(II)acetate, and Co(II)nitrate). (b) Normalized pre-edge structure after background subtraction. (c) Normalized white-line height. (d) Normalized k2 · χ(k) EXAFS spectra. in Figure 1a. The edge energy and shape for the plant sample spectra are similar to that of Co(II) and clearly different from that of Co(III). An extended and precise description of the different characteristics in the spectra of these two Co oxidation states is given by Bresson et al. (26). The small pre-edge peak (A) at 7709.5 eV, the intense so-called white 2906
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line (WL) at 7724 eV and the large bump (B) centered around 7738 eV are in agreement with an octahedral or distorted octahedral Co(II) geometry. The pre-edge feature of the sample and reference spectra was extracted by subtracting the contribution of the edge jump using an arctan function (Figure 1b). This feature is
TABLE 1. Results of the Refinement of the EXAFS Spectra Using ATHENA (XAS Normalization) and ARTEMIS (XAS sSimulation), Showing the Coordination Number with Neighboring Atoms (N), the Interatomic Distance (R), and the Debye-Waller Factor (σ2)a N
R (Å)
σ2 (Å2)
Co (C5H7O2)3 cobalt acetylacetonate
6O 6C 3C
1.89 ( 0.01 2.86 ( 0.02 3.17 ( 0.05
0.0021 ( 0.001 0.0027 ( 0.002
3.3
0.030
CoII(NO3)2.6H2O cobalt nitrate hexahydrate
6O
2.082 ( 0.009
0.0024 ( 0.0006
0.0
0.033
CoII(C2H3O2)2.4H2O cobalt acetate tetrahydrate
6O 2C 2O
2.062 ( 0.008 3.155 ( 0.02 3.21 ( 0.01
0.0064 ( 0.0005 0.0014 ( 0.0023
-5.3
0.023
CoII wheat roots
6O
2.069 ( 0.010
0.0070 ( 0.0006
1.5
0.010
CoII wheat shoots
6O
2.076 ( 0.032
0.0095 ( 0.0019
2.8
0.083
CoII tomato roots
6O
2.075 ( 0.013
0.0054 ( 0.0008
0.4
0.021
CoII tomato leaves
6O
2.068 ( 0.015
0.0054 ( 0.0009
0.4
0.025
sample III
∆E (eV)
R-factor
S02 ) 0.729 from Co metal. The R-factor is a statistical measure of the goodness of fit of the theoretical calculations to the experimental data, ∆E is the energy shift of the experimental spectra with respect to that predicted theoretically, and S02 is the factor which takes into account inelastic energy loss during the absorption process and is constant for a given edge. a
typically attributed to formal 1s to 3d transitions, a forbidden transition from a dipolar point of view but allowed due to quadrupole-dipole coupling. The pre-edge feature observed for the plant samples is at the same energy position as Co(II) and at a lower energy (-0.7 eV) than the pre-edge obtained with the Co(III) model compound. The height of the preedge feature for the plants is slightly higher than that obtained for Co(II) acetate and clearly higher than the height obtained for Co(II) nitrate. This increase can be attributed to the Co(II) in the plants being in a less centro-symmetric or distorted site than in the two Co(II) model compounds. The comparison of the normalized white line height (Figure 1c) leads to the same conclusion. The white-line corresponds to the 1s to 4p transition in the dipolar approximation. Its normalized height is approximately 1.9 for the Co(II) nitrate reference, 1.5 for Co(II) acetate and ranges between 1.54 and 1.42 for the plant samples. Such differences in the white line height are also due to an increase in Co octahedral site distortion. This has been clearly shown by Chaboy et al. (27) for Cu(II) in solution. Differences can be finally observed in the shape and position of the feature corresponding to (B) in Figure 1a. It is well marked for Co(II) nitrate, very weak for Co(II)-acetate and in an intermediate state for the plants. Concerning the Co(II) acetate spectrum, this weak feature, almost seen as a single variation of the slope of the high energy tail of the white-line, and not as a real bump, is characteristic of bonding with carboxylate groups (28). The normalized EXAFS oscillations extracted from the plant and reference samples are shown in Figure 1d. Comparison of the local structure of Co in the plant samples with the references suggests the existence of CosO or CosN binding contributions (backscattering amplitudes of the CosN and CosO bonds being almost identical). Results of the best fits for plant and reference samples are shown in Table 1. Our results for the reference compounds are in accordance with previous studies concerning the nature, numbers, and distance of the bonds around the central Co atom (28-30). As analysis of the XANES data clearly showed that Co was in an octahedral site in the plants, we fixed the number of first neighbors to 6 for corresponding simulations. Simulations were performed for the first shell using a single CosO contribution. Hypothetical CosS contributions to Co spe-
ciation in the plant samples were also examined but did not model the data with reasonable coordination parameters (data not shown). As such, it can be ascertained that in the roots and shoots of both plants, Co is most likely bound to O (or N) with a mean interatomic distance of 2.07 Å. The local order/disorder (or bond length distribution) is quantified by the σ2 value. This value is rather small for Co(II-nitrate (0.0024 Å2), slightly higher for Co(II)-acetate and the “tomato spectra” (0.0054 Å2) and clearly higher for the “wheat spectra” (0.0070 Å2 and 0.0095 Å2 for the roots and shoots, respectively). These evolutions are in complete coherence with the observations of the white line heights (Figure 1c). These differences, which are characteristic of an increase in the bond length distribution function, can be seen as proof for the complexation of Co by different organic ligands in the plants. Cobalt Distribution in Leaves. Leaf samples from both plants were randomly selected from old leaves that did not show any toxicity symptoms (e.g., necrotic spots) and were analyzed by micro-PIXE in the vicinity of the leaf mid vein. The K distribution patterns represent the vegetal structure of the wheat leaves and showed similarities to the localization of Ca (Figure 2a). Parallel veins, characteristic of monocotyledons, dominate the K map but are less obvious in the Ca map. Cobalt appeared to be distributed as the macronutrients, in particular K, with decreasing signal intensity from the vascular tissues (xylem and phloem) to the foliar limb (Figure 2a). These localization patterns were identical for both high and low dose Co exposure (data not shown). Figure 2b represents typical elemental maps of K, Ca, and Co in tomato leaves after exposure to 20 µM Co. As for the wheat leaves, the K map represents the vegetal structure. In contrast, Ca is preferentially concentrated in the trichomes. It would appear from the Co elemental map that the distribution of Co follows that of Ca. However, high concentrations of Ca can cause pulse pile-up which cannot be properly separated from the Co KR and Kβ peaks when a primitive mapping method based on energy windows is used. This problem was clearly identified by comparison of the qualitative information obtained from mapping (e.g., Figure 2b) with quantitative PIXE results determined from relevant regions on the maps, using GUPIX software (Table 2). As can be seen in Table 2, there was, in fact, no preferential sequestration of Co in the Ca-containing trichomes. The Co VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. PIXE elemental maps of a leaf sample from a wheat (a) and tomato (b) plant that had been exposed to 20 µM Co. The total collected beam charge was 3.0 µC for each sample. Elemental maps were obtained by extraction of the KsKr peak for “K”, CasKβ for “Ca” and CosKr, CosKβ for “Co” from the PIXE spectrum. The numbers of counts are increasing from light blue to yellow-green (arbitrary units). Hot spots on the Ca map of the tomato leaf represent trichomes, which contain high Ca concentrations. Note that the correlated Co hot spots with the trichomes are essentially a “false” image caused by Ca pulse-pileup (see text for details).
TABLE 2. Concentrations of K, Ca, and Co in Plant Leaves As Calculated From the PIXE Elemental Maps Using GUPIX K
Ca
Co
-1
wheat
tomato
control
whole scan
31900 ( 50
µg · g d.w. 330 ( 50
20 µM Co exposure
whole scan leaf mid vein limb near vein leaf limb
35900 ( 30 42700 ( 50 26100 ( 40 17700 ( 30
360 ( 50 400 ( 60 310 ( 40 650 ( 30
control
leaf limb scan
8750 ( 30
7240 ( 30
20 µM Co exposure
whole scan leaf mid vein leaf limb trichomes
25000 ( 40 33300 ( 70 10300 ( 30 12800 ( 80
3490 ( 40 3190 ( 60 2570 ( 20 13100 ( 70
map in Figure 2b is a false map and, therefore, not representative of Co localization. Similar results were also obtained for tomato growing in the nutrient solutions containing 5 µM Co (data not shown). The elemental concentrations of the whole scans of the wheat sample (∼300 × 300 µm2) compared to concentrations estimated in selected regions are also shown in Table 2. The results confirm that Co concentrations in the leaf veins are higher than in the averaged whole scan and also decrease from the vein to the limb (i.e., identical to K). In the tomato leaves, Co concentrations were also significantly higher in the vein than in the limb. Upon comparison between SI Tables S1 and S2 it can be noted that the Co concentrations measured by PIXE are lower than those measured by ICP-MS. We hypothesize that this is a result of using more mature leaves for the PIXE analyses, which contain less Co, than the average Co 2908
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