Biometals (2011) 24:1017–1026 DOI 10.1007/s10534-011-9459-9
Compartmentalization and ultrastructural alterations induced by chromium in aquatic macrophytes Pedro A. Mangabeira • Aluane S. Ferreira • Alex-Alan F. de Almeida • Vale´ria F. Fernandes • Emerson Lucena • Vaˆnia L. Souza • Alberto J. dos Santos Ju´nior • Arno H. Oliveira • Marie F. Grenier-Loustalot Fre´derique Barbier • Delmira C. Silva
•
Received: 17 February 2011 / Accepted: 3 May 2011 / Published online: 12 May 2011 Ó Springer Science+Business Media, LLC. 2011
Abstract The aim of the present study was to identify the sites of accumulation of Cr in the species of macrophytes that are abundant in the Cachoeira river, namely, Alternanthera philoxeroides, Borreria scabiosoides, Polygonum ferrugineum and Eichhornia crassipes. Plants were grown in nutritive solution supplemented with 0.25 and 50 mg l-1 of CrCl36H2O. Samples of plant tissues were digested with HNO3/HCl in a closed-vessel microwave system and the concentrations of Cr determined using inductively-coupled plasma mass spectrometry (ICP-MS). The ultrastructure of root, stem and leaf tissue was examined using transmission electron microscopy (TEM) and secondary ion mass spectrometry (SIMS) in order to determine the sites of accumulation of Cr and to detect possible
Electronic supplementary material The online version of this article (doi:10.1007/s10534-011-9459-9) contains supplementary material, which is available to authorized users. P. A. Mangabeira (&) A. S. Ferreira A.-A. F. de Almeida V. F. Fernandes E. Lucena V. L. Souza A. J. dos Santos Ju´nior A. H. Oliveira D. C. Silva Departamento de Cieˆncias Biolo´gicas, Centro de Microscopia Eletroˆnica, Universidade Estadual de Santa Cruz, Rodovia Ilhe´us-Itabuna km 16, Ilhe´us, Bahia 45662-900, Brazil e-mail:
[email protected] M. F. Grenier-Loustalot F. Barbier Service Central d’Analyse, CNRS, Echangeur de Solaize, Chemin du Canal, 69360 Solaize, France
alterations in cell organelles induced by the presence of the metal. Chromium accumulated principally in the roots of the four macrophytes (8.6–30 mg kg-1 dw), with much lower concentrations present in the stems and leaves (3.8–8.6 and 0.01–9.0 mg kg-1 dw, respectively). Within root tissue, Cr was present mainly in the vacuoles of parenchyma cells and cell walls of xylem and parenchyma. Alterations in the shape of the chloroplasts and nuclei were detected in A. philoxeroides and B. scabiosoides, suggesting a possible application of these aquatic plants as biomarkers from Cr contamination. Keywords Aquatic plants Heavy metals Chromium Cell ultrastructure Transmission electron microscopy Secondary ion mass spectrometry
Introduction Chromium is not essential for plants and is frequently toxic, particularly in the Cr?6 form (Shanker et al. 2005). According to Cervantes et al. (2001), plant roots are able to reduce Cr?6 to Cr?3 via a detoxification reaction mediated by chromium reductases, and this could form the basis of a phytoremediation process for toxic Cr?6. In this context, phytoremediation comprises the removal by detoxification or stabilization of contaminating metals in soils and water systems by plants. Thus, high levels of metals that
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are normally toxic to other organisms may be tolerated by plants that can absorb the pollutants through the roots and either detoxify them or translocate them directly to the aerial parts where they are stored at very high concentrations (so-called hyperaccumulation) (Mishra and Tripathi 2009). In this manner, phytoremediation allows a full or partial decontamination of soils whilst preserving their biological activities, and provides an attractive alternative to other remediation processes involving the immobilisation or extraction of metal contaminants, which are both expensive to apply and limited to small areas (Baker et al. 1991). Furthermore, phytoremediation is an environmentally friendly technology that offers the possibility of in situ recovery of metals for further use (Pilon-Smits 2005). By virtue of its low-cost, phytoremediation has been targeted by governmental agencies and private enterprises with limited budgets. It has been demonstrated that aquatic macrophytes have the potential to remove pollutants and can be used as biomarkers for the occurrence of heavy metals in aquatic environments (Maine et al. 2001). Metal accumulation in macrophytes is frequently accompanied by cell modifications that may contribute to their metal tolerance (Prasad and Freitas 2003). Investigations involving high resolution ion microscopy are scarce in biology (Jauneau et al. 1992; Grignon et al. 1996; Mangabeira et al. 2004), although the technique can be used together with transmission electron microscopy (TEM) for the precise detection of heavy metals in plant tissues and cells. Thus, the location of the metal in the sample can be determined by comparison of ion secondary image (ISI) generated by secondary ion mass spectrometry (SIMS) with the image of the element under investigation (in the present case Cr). The aim of the present study was to employ TEM and SIMS in order to identify the sites of accumulation of Cr in the species of macrophytes that are abundant in the Cachoeira river, namely, Alternanthera philoxeroides (Mart.) Griseb., Borreria scabiosoides Cham. and Schltdl., Polygonum ferrugineum Wedd. and Eichhornia crassipes (Mart.) Solms. The capacity of each macrophyte for phytoremediation was assessed on the basis of accumulation of Cr in tissues and cells, and the ultrastructure of root, stem and leaf tissue was examined in order to evaluate the effect of Cr and to detect possible alterations in the cell organelles.
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Materials and methods Plant material and cultivation conditions The macrophytes A. philoxeroides, B. scabiosoides, P. ferrugineum and E. crassipes were collected from Cachoeira river at locations in uncontaminated area near to Ilhe´us (14°470 –14°480 S; 39°060 –39°080 W), and transported in plastic buckets containing water to the Centro de Pesquisa do Cacau (CEPEC) in Ilhe´us, Bahia, Brazil. The experiment was conduced in greenhouse (14°470 S 39°160 W 55 m a.s.l.) at the follow conditions 30% of the total incidence radiation, temperature of 23 ± 1°C, relative humidity of 84 ± 3%. Plants were then transferred to 30 l trays containing 1/4 strength Hoagland and Arnon (1950) nutrient solution and allowed to acclimate for a period of 30 days. After this time, some plants were transferred to nutrient solution supplemented with CrCl3 6H2O (25 or 50 mg l-1) and others were maintained in nutrient solution without Cr. Incubation was carried out under constant aeration for 90 days, during which time the level of nutrient solution within each tray was maintained by adding deionised water, and the pH was monitored and adjusted to 5.8 with NaOH or HCl on a daily basis. The nutrient solution was replaced every 15 days. Chemical analysis The plants were separated into roots, stems and leaves, except for E. crassipes which was separated into roots and shoots. Samples (50 mg dry weight) of plant material were digested with analytical grade HNO3 (4.0 ml) and HCl (1.0 ml) in closed Teflon digestion bombs heated to 190°C for 12 min in a 1,000 W Ethos Plus Microwave Labstation (Milestone Inc., Monroe, CT, USA). After cooling, the concentrations of Cr in the samples were determined using an inductivelycoupled plasma mass spectrometer (ICP-MS) model PQ3 Excel (Thermo Scientific, Waltham, MA, USA). Signal drift due to matrix effects was monitored by adding internal standard (10 lg l-1 of Cr) to both sample and standard solutions according to the established protocol of the manufacturer. All estimations were conduced in triplicate and the concentrations were expressed in mg per kg dry weight. The overall recovery associated with the digestion process was found to be in the range 87–95%.
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TEM analysis TEM analyses were carried out in the Electron Microscopy Center of the Universidade Estadual de Santa Cruz, Ilhe´us, BA, Brazil. In order to avoid confusion between stained cell structures and sites of Cr accumulation, TEM slides were not stained with uranyl acetate and lead citrate. Root, stem and leaf tissues were immersed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 6.9, cut into small fragments (ca. 1 mm3), submitted to a weak vacuum for 30 min and subsequently maintained under normal pressure for a further 1 h. Samples were then submitted to six washes (10 min each) with sodium cacodylate buffer, fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 4 h at 4°C, washed six times (10 min each) with sodium cacodylate buffer, and dehydrated in an ethanol gradient (30, 50, 75, 85 and 95% ethanol, followed by three washes in 100% ethanol). Finally, samples were covered sequentially with solutions of ethanol:epoxy resin (Spurr 1969) in proportions of 3:1, 1:1 and 1:3, and then three times with pure Spurr resin, the final treatment being continued overnight at room temperature. Next day, samples were placed in silicon moulds, covered with pure Spurr resin and polymerised overnight. The polymerised resin blocks were trimmed with a razor blade, thinly sectioned (2 lm) with a glass blade, and ultra-thinly sectioned (60–70 nm) with a diamond blade using a ultramicrotome (model EM FC6 LEICA Microsystems). Thinly-cut sections were placed between glass slides and cover slips for structural examination. Ultra-thin sections were cut and placed onto copper mesh grids and examined using a MorgagniTM 268D TEM (FEI Company, Hillsboro, OR, USA) equipped with a CCD camera and controlled by software running under Windows OS. High-resolution SIMS analysis Samples were rapidly frozen by plunging into liquid propane cooled to -196°C with liquid nitrogen, transferred to a cryogenic vessel (model EM-AFS, Leica Microsystems, Wetzlar, Germany) containing 20 ml of acetone pre-cooled to -92°C, and left for 1 week. Frozen specimens were embedded in Lowcryl K4M resin, polymerised at -20°C for 2 days, cut into 2 lm sections using a Reichert-Jung Ultracut E
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ultramicrotome and placed either on a square gold plate for analysis by SIMS or between glass slides and cover slips for correlation with light microscopy. High resolution SIMS analyses were performed at the Division of Biological Sciences, Department of Medicine, Enrico Fermi Institute, University Chicago, IL, USA. A Finnigan MAT 90 magnetic sector mass spectrometer (Thermo Scientific, Waltham, MA, USA) was combined with a scanning ion microprobe (Strick et al. 2001), by which the samples were illuminated with a gallium focused 40 nm ion beam in order to obtain high lateral resolution massresolved images of their surfaces. Secondary ions were detected using an ETP AF820 active film electron multiplier (Scientific Instruments Services, Ringoes, NJ, USA) operating in the pulse counting mode at count rates up to 50 MHz. In order to locate Cr, samples were scanned in the positive mode with the 52Cr? SIMS signal displayed on a CRT at fields of view 80 and 160 lm wide. SIMS images (512 9 512 pixels) formed by single square raster scans were stored and analysed using Kontron IMCO imaging system (Kontron, Fremont, CA, USA). Experimental design and statistical analysis The experiment followed a randomised design and involved the treatment of plants with two different concentrations of Cr (25 and 50 mg l-1) with five repetitions each. The mean concentrations of Cr accumulated in root, stem and leaf tissues were compared by analysis of variance and the Tukey test (q \ 0.05).
Results Accumulation of Cr in plant tissues ICP-MS analyses of plant tissues demonstrated that Cr accumulated mainly in the roots of the macrophytes, with significantly (q \ 0.05) smaller levels in the stems and, with the exception of E. crassipes, almost negligible amounts in the leaves (Table 1). Moreover, the levels of accumulation of the metal in the roots of A. philoxeroides, P. ferrugineum and E. crassipes differed significantly (q \ 0.05) according to the treatment applied (i.e. 25 or 50 mg l-1 of Cr). The largest accumulations of Cr were observed in the roots
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of A. philoxeroides that had been grown in 50 mg l-1 of Cr. A maximum accumulation (29.16 mg Cr kg-1 dw) of Cr was observed in the roots of A. philoxeroides that had been grown in the presence of 50 mg l-1 of CrCl3, although high levels (ca. 29.16 mg Cr kg-1 dw) were also found in the roots of P. ferrugineum and B. scabiosoides grown under similar conditions. The total uptake of Cr into the roots appeared to reflect the concentration of metal in which the plant was grown. However, the total Cr accumulation in leaf tissue, which was generally insignificant, was not proportional to the treatment concentrations indicating that translocation of the metal from roots to leaves/shoots was a rate-limiting step.
Values represent the mean of three repetitions ± standard error
Effects of Cr treatment on the ultrastructure of plant tissues as determined by TEM and SIMS Values bearing dissimilar upper case (columns) or lower case (rows) letters are significantly different according to Tukey test (q \ 0.05)
29.16 ± 0.480Ca 5.75 ± 0.081Bb 0.15 ± 0.004Bc 19.07 ± 0.081Ba 4.94 ± 0.334Bb 0.13 ± 0.002Bc 19.48 ± 0.08Ca 50
8.66 ± 0.163Bb 0.04 ± 0.001Bc 17.75 ± 0.668Ca 9.02 ± 0.033Bb
0.01 ± 0.001Aa 0.01 ± 0.001Aa 0.02 ± 0.001Aa 0.01 ± 0.001Aa 0.01 ± 0.001Aa
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0.02 ± 0.001Aa 0.01 ± 0.002Aa 0.01 ± 0.001Aa 0.02 ± 0.001Aa 0.01 ± 0.001Aa 0.01 ± 0.001Aa
21.18 ± 0.192Ba 4.54 ± 0.039Bb 0.10 ± 0.003Bc 13.70 ± 0.204Ba 3.78 ± 0.033Bb 0.10 ± 0.001Bc
0
Roots Roots
Stems
Leaves
Roots
Stems
Leaves
Roots
Stems
Leaves
E. crassipes (mg Cr kg-1 dw) P. ferrugineum (mg Cr kg-1 dw) B. scabiosoides (mg Cr kg-1 dw) CrCl36H2O A. philoxeroides (mg Cr kg-1 dw) (mg l-1)
Table 1 Concentrations of Cr in tissues of macrophytes cultivated for 90 days in nutrient solutions containing different amounts of CrCl36H2O
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8.61 ± 0.114Ba 5.25 ± 0.166Bb 0.02 ± 0.004Bc 10.91 ± 0.429Ba 4.68 ± 0.053Bb
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Shoots
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Alterations in the ultrastructure of leaves, stems and roots of A. philoxeroides that had been grown in the presence of Cr could be readily detected by TEM (Fig. 1) and SIMS (Fig. 2) analysis. The presence of Cr at 50 mg l-1 induced the most severe modifications, which included changes in nuclear shape and envelop integrity together with modifications in the shape of leaf chloroplasts, resulting in the structural disarrangement of thylakoids and stroma in comparison with control plants. Modifications in cell ultrastructure detected by TEM analyses of leaves, stems and roots of B. scabiosoides, P. ferrugineum and E. crassipes that had been incubated in 50 mg l-1 of Cr are shown in Fig. 3 and Supplementary Figs. 5 and 7, respectively, while those revealed by SIMS analysis are presented in Fig. 4 and Supplementary Fig. 6, respectively. Of particular note were the modifications in the mitochondrial cristae induced by Cr in root cells of B. scabiosoides (Fig. 3i).
Discussion Of all of the species and plant parts analyzed, the roots of A. philoxeroides exhibited the highest concentration of Cr. Naqvi and Rizvi (2000) reported significant accumulations of Cr in the roots of A. philoxeroides. In the case of E. crassipes, while the roots were the preferential site of metal accumulation, with the cell walls and vacuoles accumulating high levels of Cr, there was a modest translocation to the aerial parts
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Fig. 1 TEM electron micrographs of transversal sections of leaf, stem and root tissue of A. philoxeroides following treatment of plants with 50 mg l-1 Cr: a electron-dense material (arrowed) in cell wall (cw) of leaf, bar 2 lm; b deformed chloroplast (cl) and nucleus (n) in leaf cell, bar 2 lm; c Cr deposits (arrowed) in the cell wall (cw) of stem
xylem, bar 5 lm; d Cr deposits in the cell wall (cw) of stem parenchyma, bar 5 lm; e deformed chloroplast (cl) and nucleus (n) in stem cells, chromium deposits (arrows), bars 2 lm; f disintegration of nucleus (n) in root cell (arrow), bars 1 lm
particularly when the treatment involved a high concentration of Cr (Table 1). Other authors (Maine et al. 2001; Ingole and Bhole 2003; Paiva et al. 2009) have also described the greater accumulation of Cr in roots of E. crassipes in comparison with the aerial parts, whilst Mishra and Tripathi (2008) demonstrated that E. crassipes was more efficient in the removal of heavy metals than Pistia stratiotes or Spirodela polyrrhiza. On the other hand, Mang et al. (2008) recently reported that Cr absorption by A. philoxeroides
roots was greater than by roots of E. crassipes, a finding that is agreement with the results of the present study. These researchers employed Fourier transform infrared (FTIR) spectrometry to show that the root cell walls of Cr-treated plants exhibited a significant shift in –OH absorption peaks in comparison with control plants, and suggested that –OH and COO– groups were associated with Cr binding in aqueous solutions and that this might constitute a mechanism of Cr accumulation.
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1022 Fig. 2 SIMS images of stem and root tissue of A. philoxeroides following treatment of plants with 50 mg l-1 Cr: a ISI of stem parenchyma (p); b Cr deposits in cell wall and vacuole of stem parenchyma (p) at a depth of 20 nm; c ISI of the vessel element (ve) and parenchyma (p) of stem xylem; d Cr deposits in cell wall of the vessel element of stem at a depth of 20 nm; e ISI of vessel element (ve) of root xylem; f Cr deposits (arrowed) in the vessel element (ve) of root xylem at a depth of 20 nm (arrow); g ISI of root parenchyma; h Cr deposits in the cell wall (cw) and vacuole (v) of root parenchyma at a depth of 20 nm
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Fig. 3 TEM electron micrographs of transversal sections of leaf, stem and root tissue of B. scabiosoides following treatment of plants with 50 mg l-1 Cr: a Deformed chloroplast (arrows) in leaf cell, bar 7 lm; b normal chloroplast with thylakoid and starch granule (sg) in control leaf, bar 1 lm; c deformed chloroplast (cl; arrowed) in leaf cell, bar 5 lm; d normal nucleus (n) in control leaf, cl (chloroplast), bar 5 lm;
e deformed nucleus (n) in leaf cell, bar 5 lm; f Cr deposits (arrowed) in stem cell wall (cw), bar 2 lm; g Cr deposits (arrowed) in the cell wall and cell wall of stem xylem, bar 5 lm; h Cr deposit (arrowed) in cell wall (cw) and vacuole of root cells, bar 1 lm; i electron dense material (arrowed) in mitochondria (m), alterations in mitochondrial cristae (asterisk) and Cr deposit in vacuole (v) in root cells, bar 1 lm
The results of the present study clearly indicate that Cr is strongly adsorbed to the cell walls of the roots and that translocation to the aerial parts is negligible, as described in an earlier report (Pulford and Watson 2003). It is possible, however, that part of the metal taken up by the roots may cross the plasma membrane and become bound to macromolecules, organic acids, or sulphur-rich polypeptides, such as phytochelatins, thereby accumulating in the cytoplasm or the vacuole and becoming detoxified (Harmens et al. 1994). Indeed, it has been suggested that the formation of complexes between Cr and organic acids may play an important role in the inhibitor/stimulator effect of Cr
on the translocation of different minerals (Panda and Choudhury 2005). Mangabeira et al. (2004) employed ion microscopy to detect large amounts of Cr in the vascular cylinder of E. crassipes roots and stems, particularly around the secondary xylem. In addition to the presence of Cr in the root parenchyma, these authors observed Cr in the transport parenchyma, indicating that such cells were responsible for conveying the metal to the leaves. It was concluded that compartmentalisation of Cr in the vacuoles was important for the detoxification and tolerance of macrophytes towards the metal. Srivastava et al. (1999) proposed
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Fig. 4 SIMS images of stem and root tissue of B. scabiosoides following treatment of plants with 50 mg l-1 Cr: a ISI of stem parenchyma (p); b Cr deposits in stem parenchyma (p) at a
depth of 20 nm; c ISI of the root xylem (x); vessel element (ve); d Cr deposits in vessel element (ve) of root xylem (x) and parenchyma cell wall (p) at a depth of 20 nm
that Cr forms metal chelates with organic acids within the vacuoles or small cell vesicles, which are responsible for the detoxification and tolerance to metal stress. On the basis of such evidence, it is possible to state that the macrophyte species studied are, to some extent, tolerant to Cr, since they are capable of accumulating the metal in vacuoles. Other floating macrophytes including A. sessilis, Salvinia herzogii and P. stratiotes also accumulate large amounts of Cr in the roots, and it has been shown that in such plants the metal tends to be immobilised in the radicular system in order to limit aerial toxicity (Sinha et al. 2002; Maine et al. 2004). In this context, Mishra and Tripathi (2009) explained the accumulation of heavy metals in the roots of macrophytes in terms of an active uptake of heavy metals by plasmolysed cells and the impregnation of cell walls
via passive diffusion. However, a different explanation has been given by Barbosa et al. (2007) who stressed the tendency of metal ions to either bind to other ions or precipitate within the cell walls, thus preventing the translocation of the metal to the aerial parts. These authors have emphasised that Cr is not only toxic but is also unnecessary for the development of plants, thus accounting for the absence of specific mechanisms for Cr transport from roots to leaves. Panda and Choudhury (2005) suggested that Cr-induced oxidative stress results in the peroxidation of membrane lipids, causing severe damage to the cell membranes and degradation of photosynthetic pigments. These authors also claimed that high concentrations of Cr may cause damage to chloroplast ultrastructure and affect photosynthesis. Since Cu, Fe and Cr present different redox potentials, their
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influence regarding induction of oxidative stress exceeds that of other metals such as Co, Zn and Ni. Moreover, Cr toxicity has been attributed to the interference by the metal in photosynthetic electron transport (Larcher 1995). On this basis, it is possible that Cr may have exerted an effect on the photosynthetic processes in the aquatic macrophytes studied herein, possibly negatively influencing the growth and development of these species. However, in the present study no alterations were observed in the chloroplasts of P. ferrugineum or E. crassipes following treatment of the plants with 50 mg l-1 Cr. In contrast, Lage-Pinto et al. (2008) reported structural changes, including thylakoid disorganisation, in the chloroplasts of samples of E. crassipes originating from industrial areas. Indeed, according to Panda and Choudhury (2005), alterations in chloroplast shape induced by biotic and abiotic stress result from an increase in stroma volume and disorganisation of thylakoids. Alternanthera philoxeroides and B. scabiosoides that had been treated with 50 mg l-1 Cr presented alterations in cell nuclei, including disintegration of the nucleus, suggesting that high concentrations of Cr may lead to cell death. Similar findings have been reported by Rocchetta et al. (2007) following the analysis of Cr-treated Euglena gracilis. Additionally, roots of B. scabiosoides exhibited electron dense deposits in the cell walls together with alterations in mitochondrial cristae, in agreement with results reported for Cr- and Ni-treated Allium cepa (Liu and Kottke 2003). The occurrence of Cr deposits in the cell walls of root parenchyma and in the xylem vessel elements of the macrophytes studied may be explained by the slow diffusion of the metal together with cation exchange at specific sites in the roots cells (Shanker et al. 2005). In addition, the occurrence of Cr deposits in the stem xylem of A. philoxeroides, B. scabiosoides and E. crassipes could be due to active uptake of Cr by plasmolysed cells and impregnation of cell walls via passive diffusion (Zayed and Terry 2002). In this context, metallothioneins and organic acids are important components of the cells walls of the secondary xylem that contribute to the tolerance and detoxification of Cr. For example, cell walls contain large amounts of polygalacturonic acids, the ionised groups of which are located on the external surface of the cells and can bind to cations
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(Grignon et al. 1996). According to the evidence revealed by the profiles of cationic abundance in plant tissues, such cations are linked to structural polymers or may be precipitated with pyroantimoniate (Jauneau et al. 1992; Ripoll et al. 1993). Acknowledgments The authors wish to thanks Dr. Lionel Dutruch (Service Central d’Analyse, CNRS), and Drs. Ricardo Levi-Setti and Konstantin Gavrilov (Enrico Fermi Institute, University of Chicago) for their kind assistance with ICP-MS and ion microscopy imaging. This research was supported by CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Brazil).
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