Spatial Imaging and Speciation of Lead in the Accumulator Plant

Jul 7, 2010 - Davis, California 95616, Stanford Synchrotron Radiation. Lightsource, SLAC National Accelerator Laboratory, Menlo. Park, California 9402...
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Environ. Sci. Technol. 2010, 44, 5920–5926

Spatial Imaging and Speciation of Lead in the Accumulator Plant Sedum alfredii by Microscopically Focused Synchrotron X-ray Investigation SHENGKE TIAN,† LINGLI LU,† X I A O E Y A N G , * ,† S A M U E L M . W E B B , § YONGHUA DU,| AND P A T R I C K H . B R O W N * ,‡ MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Science, Zhejiang University, Hangzhou 310029, China, Department of Plant Sciences, University of California, Davis, California 95616, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, and Institute of Chemical & Engineering Sciences, Agency for Science, Technology and Research (ASTAR), Singapore

Received December 24, 2009. Revised manuscript received June 10, 2010. Accepted June 23, 2010.

Sedum alfredii (Crassulaceae), a species native to China, has been characterized as a Zn/Cd cohyperaccumulator and Pb accumulator though the mechanisms of metal tolerance and accumulation are largely unknown. Here, the spatial distribution and speciation of Pb in tissues of the accumulator plant was investigated using synchrotron-based X-ray microfluorescence and powder Extended X-ray absorption fine structure (EXAFS) spectroscopy. Lead was predominantly restricted to the vascular bundles of both leaf and stem of the accumulator. Micro-XRF analysis revealed that Pb distributed predominantly within the areas of vascular bundles, and a positive correlation between the distribution patterns of S and Pb was observed. The dominant chemical form of Pb (>60%) in tissues of both accumulating (AE) and nonaccumulating ecotype (NAE) S. alfredii was similar to prepared Pb-cell wall compounds. However, the percentage of the Pb-cell wall complex is lower in the stem and leaf of AE, and a small amount of Pb appeared to be associated with SHcompounds. These results suggested a very low mobility of Pb out of vascular bundles, and that the metal is largely retained in the cell walls during transportation in plants of S. alfredii.

Introduction Heavy metal pollution is a widespread and important environmental concern. Various in situ and ex situ cleanup * Corresponding author phone: +86-571-86971907; fax: +86-57186971907; e-mail: [email protected] or [email protected]. Corresponding author address: College of Natural Resources and Environ Science, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China (X.Y). Corresponding author phone: (530)752-0929; e-mail: [email protected]. Corresponding author address: Department of Plant Sciences, University of California, Davis, CA 95616 (P.H.B.). † Zhejiang University. § Stanford Synchrotron Radiation Lightsource. | Institute of Chemical & Engineering Sciences. ‡ University of California. 5920

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technologies have been employed; of these methods, phytoremediation is promising due to its low implementation costs and significant environmental benefits (1, 2). Hyperaccumulator and accumulator species, which efficiently tolerate and accumulate heavy metals from the soil into shoots, are of potential use in the development of phytoremediation technologies for contaminated environments (3). Understanding the mechanisms of metal tolerance and accumulation will provide insight into the identification and management of these hyperaccumulating species. To date, most studies on mechanisms of metal uptake, transport, accumulation, and tolerance in plants have been performed on the Ni, Cd, or Zn hyperaccumulating species, most belonging to the Brassica family (4, 5). Comparatively fewer studies have been conducted on the accumulation mechanisms of Pb, one of the most serious pollutants that affect plant productivity and causes health hazards to animals and man. It has been reported that most of the Pb taken up by plants is restricted to roots and only a very small amount is transported to the shoots (6, 7), while the presence of chelating agents, such as EDTA, was able to facilitate the uptake and transport of Pb into the plants (8). The mechanism of plant tolerance to Pb, however, is largely unknown. Sedum alfredii Hance (Crassulaceae) is a recently identified Zn/Cd cohyperaccumulator native to the Pb/Zn rich regions of China (9–12). This species also exhibits a strong ability to both tolerate and accumulate considerable amounts of Pb (13). Preliminary studies have demonstrated that the accumulating ecotype (AE) of S. alfredii, collected from contaminated sites (Figure S1), can grow well at 100 µM soluble Pb and accumulate 4-fold higher Pb content in the aerial parts than the nonaccumulating ecotype (NAE) grown under the same conditions (Figure S2). A better understanding of the biological mechanism involved in Pb accumulation in this species would help to improve the knowledge of Pb tolerance in plants. Clemens (14) has hypothesized that chelation and sequestration processes could remove the toxic ions from sensitive sites, resulting in their effective detoxification within plants. The aim of this study was to obtain fundamental information on Pb accumulation and tolerance mechanisms in S. alfredii, by investigation of the metal localization and speciation. In recent years, the utilities of synchrotron-based techniques such as X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) for biological samples has improved dramatically, and it is now possible to conduct localization and chemical speciation studies of metals or metalloids with minimal sample preparation (15–18). While the distributions of Cd and Se in the hyperaccumulators has been successfully analyzed by Micro-XRF (15, 16), much less information is available on the metal Pb. The technique of XAS has been applied to investigate Pb speciation in soils, sediments, and earthworms in recent years (19–21) as well as the plant samples (22–25). A combination of µ-XRF and Pb LIII-edge Extended X-ray absorption fine structure (EXAFS) spectroscopy represent promising tools to study metal storage and trafficking mechanisms in plants and other biological samples. In the present study, the spatial distribution and speciation of Pb in leaf and stem tissues of S. alfredii was studied using µ-XRF imaging and EXAFS.

Materials and Methods Plant Culture and Treatments. The accumulator ecotype (AE) of S. alfredii was originally obtained from an old Pb/Zn mine area in Zhejiang Province, China, and the nonaccumulator ecotype (NAE) of S. alfredii from a tea plantation in 10.1021/es903921t

 2010 American Chemical Society

Published on Web 07/07/2010

Hangzhou in Zhejiang Province. Parent plants were grown in noncontaminated soil for several generations to reduce internal metal contents, and then uniform and healthy shoots were cut and cultivated in the basal nutrient solution containing (mM) the following: 2 KNO3, 0.05 KCl, 0.5 Ca(NO3)2, 0.2 MgSO4, 0.1 NH4NO3, 0.01 KH2PO4, 0.012 H3BO3, 0.002 MnSO4, 5 × 10-4 ZnSO4, 2 × 10-4 CuSO4, 1 × 10-4 Na2MoO4, 1 × 10-4 NiSO4, 0.02 Fe-EDTA. Nutrient solution pH was adjusted daily to 5.8 with 0.1 M NaOH or HCl. Rooted cuttings of the two S. alfredii ecotypes were precultured for 4 weeks before exposure to 100 µM Pb levels. Each treatment was replicated three times. Plants were harvested after Pb exposure for 30 d. All plants were grown under glasshouse conditions with natural light, day/night temperature of 26/ 20 °C and day/night humidity of 70/85%. The nutrient solution was continuously aerated and renewed every 3 d. µ-XRF. Sample Preparation. Stems and leaves were cut from plants grown as described above after 30 d exposure to 100 µM Pb and were surface rinsed. The midtransverse areas of stem and mature leaf samples at similar developmental stages were selected from both ecotypes for comparisons. Sections (100 µm thick) of samples were cut with a cryotome (LEICA, CM1850) at a temperature of -20 °C. Briefly, stem and leaf samples were frozen by liquid nitrogen and fixed immediately on the specimen disks using deionized water at the actively cooled (-40 °C) specimen quick freezing shelf. After about 10 min, samples were subjected for sections at a temperature of -20 °C. Sections in good conditions were selected and freeze-dried under -20 °C for 3 d. µ-XRF Analysis. Micro XRF imaging of Pb in the leaf cross sections of the plants was carried out on beamline 4A at the Photon Factory (PF), High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. Instruments and measurement conditions were modified only slightly from those described by Tian et al. (26). The electron energy in the storage ring was 2.5 GeV with a current range from 300 to 420 mA and the detector was Si(Li) solid-state detector (Princeton Gamma-Tech, Instruments Inc.). The incident beam was monochromatized by a W/B4C double multilayer monochromator to achieve a higher intensity (ca. 1010 photons s-1). The brilliance of the beam in BL-4A, KEK was 3.31 × 1014 photons/s/mm2/mrad2/0.1%BW. The beam was focused by K-B optics to 3.5* 5.5 µm. K-B optics, invented by Kirkpatrick-Baez, consists of a horizontal focusing mirror (HFM) and vertical focusing mirror (VFM), three-stripe (Rh, Ni, and Au) mirrors on polished ultralow-expansion (ULE) substrates with benders and located in the first optical enclose, after the monochromator. This device provides both very high accurace in the beam focusing and flexibility. The surface of the K-B mirror used here was coated with Rh, consequently, the intensity obtained from the optic around 14 KeV was 4-5 times higher than that obtained by K-B mirror coated with Pt, which had been previously used in this beamline. By adjusting the distance between the detector and the sample, the dead time was also controlled and kept below 30% throughout the analysis in order to reduce counting loss. The step size was set to 7 µm. Dwell time per point was 1 s. The spectra were analyzed by AXIL. The µ-XRF analysis of Pb on stem of AE S. alfredii was carried out on Stanford Synchrotron Radiation Laboratory (SSRL) beamline 2-3 using a Si (220) double-crystal monochromator with harmonic rejection achieved by detuning one monochromator crystal to 50% peak intensity, with the storage ring Stanford Positron Electron Accelerating Ring (SPEAR) containing 90-100 mA at 3.0 GeV. The brilliance of the beam in BL2-3 was 1 × 1012 photons/s/mm2/mrad2/ 0.1%BW. The microfocused beam of 2 µm was provided by a Kirkpatrick-Baez mirror pair (Xradia Inc.) with the sample at 45° to the incident X-ray beam. The fluorescence yield was detected using a single channel Vortex Si detector. The

elemental distribution maps were collected using a 5 µm pixel size. Dwell time per point was 200 ms. Beamline 2-3 saves full XRF spectrum data at each pixel. Elemental distributions are achieved by windowing on the elements of interest in the XRF spectra. The windows can be applied during data collection or during data analysis since the full XRF spectrum is saved for each pixel. The beam energy was set to 15 Kev during mapping. The fluorescence energies windowed for this investigation were P, S, K, Ca, Zn, Fe, and Pb. It is noteworthy that the Pb M lines and S K lines overlap significantly (2345 and 2308). The contribution of Pb in the S ROI window was subtracted based on the ratio of the relative intensities of the L and M emission lines from a pure Pb spectra. The elemental maps focused on the vascular bundles were collected using a 2 µm pixel size, under the same conditions. The fluorescence data are presented as tricolor maps that allow for the spatial distribution of three elements to be shown. Pixel brightness is displayed in RGB, with the brightest spots corresponding to the highest element fluorescence. The maps were produced using the software package SMAK version 0.34, S-4 (http://www-ssrl.slac. stanford.edu/∼swebb/smak.htm). Bulk EXAFS. EXAFS data of powdered tissues (roots, stems, and leaves) from the S. alfredii plants were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) with the storage ring SPEAR-3 operating at 3 GeV and with ring currents of 80-100 mA. Lead LIII-edge spectra were recorded on beamline 7-3 with an upstream Rh-coated collimating mirror, a Si(220) double-crystal monochromator, and a downstream focusing mirror. The incident X-ray intensity was monitored using a Kr filled ionization chamber. The monochromator energy of each spectrum was calibrated using Pb metal foil between the second and third ionization chambers; its absorption edge was calibrated to an edge of 13035 eV. Pb LR fluorescence were recorded using a 30 element germanium detector (Canberra Industries, Meriden, CT) equipped with Soller slits and selenium filters. Fresh plant tissues were ground under liquid nitrogen and pressed into 2-mm path length Lucite sample holders with Kapton tape windows cooled in liquid nitrogen. To minimize breakdown and mixing of cellular components within the plant material, care was taken to keep the tissue frozen at all times during measurement. During data collection, samples were maintained approximately at 10 K in a liquid helium flow cryostat to minimize the loss of intensity in the signal. Pb at room temperature gives a high signal/ noise ratio because its Debye temperature is very low; samples were kept at the low temperature to avoid this and to minimize the oscillations of the low Z Pb ligands, such as O and C. Spectra were also collected of standard lead species including Pb3(PO4)2 (solid), Pb(CH3COO)2 (solid), Pb(NO3)2 (solution), Pb-malate (solution), Pb-glutathione (solution), Pb-citrate (solution), and Pb-cell wall (solid). All solution was prepared in 30% glycerol to prevent ice crystal formation. The complexes of Pb-malate, Pb-citrate, and Pb-glutathione were made by adding 5.0 mM citrate, malate, or glutathione to an aqueous solution of 0.5 mM Pb(NO3)2, pH 6. Root cell wall material was exposed to 0.5 mM Pb(NO3)2 in hydroponic solution (final pH ) 6) for 72 h, rinsed in deionized water, and frozen in liquid nitrogen for storage. Spectra of all plant samples and stand solution samples were recorded in fluorescence mode, while spectra of solid stand samples were recorded in transmission mode. Detuning of the primary beam by 50% was performed to reject higher harmonic reflections. EXAFS Data Analysis. Multiple scans (4-16 depending on Pb concentration) were collected and averaged for each sample to improve the signal-to-noise ratio. The spectra data of each sample were calibrated to the lead LIII-edge (13035 eV) in the program SixPack (27). The normalization of the VOL. 44, NO. 15, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. µ-XRF elemental maps for Pb, Ca, K, Zn, P, and S of leaf cross sections from AE (a) and NAE (b) S. alfredii treated with 100 µM Pb for 30 d. The number of fluorescence yield counts was normalized by I0 and the dwell time. The red color, depicting elemental concentrations in each map, was scaled to the maximum value for each map. UE, upper epidermis; LE, lower epidermis; SM, spongy mesophyll; PM, palisade mesophyll; V, vein. Scale bar: 100 µm. EXAFS spectra was carried out according to standard methods using the SixPack program suite, and the spectra were normalized to unit step height using a linear pre- and postedge background subtraction. The spectra were transformed to k-space based on E0 equal to 13035 eV. The k-function was extracted from the raw data by subtracting the atomic background using a cubic-spline consisting of 7 knots set at equal distances fit to k3-weighted data; k3weighted (k) functions were Fourier transformed over the 2 to 10 Å-1 range using a Kaiser-Bessel window with a smoothing parameter of 4. The k3-weighted EXAFS spectra recorded on the plants were least-squares fitted over a wave vector (k) range of 1.5-10 Å-1 using a combination of Pb compounds standard. Best fits were derived by incrementally increasing the number of fit components and minimizing the fit residual. Fits were optimized by minimizing the residual of the fit, defined as the normalized root square difference between the data and the fit. The range for the fit was varied as a function of data quality and in order to test contributions from minor components. Further details of fitting procedures can be found in refs 24, 28, and 29.

Results Spatial Imaging of Pb and Other Elements in Leaf and Stem. To investigate the spatial distribution of Pb in the accumulator plant, micro scanning XRF mapping was performed on both AE and NAE S. alfredii. The X-ray fluorescence spectrum from the epidermis of a leaf cross5922

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section from AE S. alfredii showed clear peaks of K, Ca, Mn, Fe, Cu, Zn, P, S, and Pb (Figure S3). The integrated intensity for each element was calculated from the spectrum and normalized by the intensity of the Compton scattering peak. Elemental mapping for the measurement area was obtained from the normalized intensity for each element. The elemental distribution maps of Pb, Ca, K, Zn, P, and S in the scanned area of leaf and stem cross sections are presented in Figures 1 and 2, respectively, together with photographs taken using an optical microscope. Each map indicates the relative distribution of the specific element, and the counts scales vary for each map. The normalized X-ray fluorescence intensities are scaled to red (maximum) and to blue (minimum) for individual elements. The leaf cross-section image of AE (Figure 1a) revealed that Pb was accumulated mainly in the leaf veins, with a second peak in the epidermis. A much lower content of Pb was found in either spongy or palisade mesophyll cells (Figure 1a). Distribution patterns of S in the leaf cross-section were quite similar to that of Pb (Figure 1a), and a positive correlation between the XRF intensities of Pb and S was observed (correlation coefficient: r2 ) 0.514, data number: n ) 11210, p < 0.001) (Figure S4). No marked concentration of other elements (Ca, K, P, and Zn) in leaf veins was observed (Figure 1a). Phosphorus was evenly distributed in the leaf cross sections of AE, and relative higher levels of K were located in spongy tissues, while Ca was concentrated in palisade tissues. Preferential localization of Zn in epidermis

FIGURE 2. µ-XRF elemental maps for Ca (c), Pb (d), K (e), Zn (f), P (g), and S (h) of stem cross sections from AE S. alfredii treated with 100 µM Pb for 30 d. (a) is the photograph of the measured samples. Scale bar: 100 µm. (b) shows the intensity of Pb in the scanned sites from point A to point B marked in Figure 2 (d). Beam size, 2*2 µm; step size, 5 µm. was observed, while the lowest level of this element in a leaf cross-section was observed in vascular tissues. In NAE leaves (Figure 1b), Pb was the most concentrated in the vascular bundles, whereas Ca, P, and S were uniformly distributed in the cross sections of leaf. Distribution patterns of K were weakly correlated with Pb. Zinc in leaves of NAE plants was also preferentially localized in epidermis. No significant correlation between intensity of Pb and other elements was observed in the leaf cross sections of NAE plants (data not shown). Micro-XRF elemental mapping of stem cross sections of the AE S. alfredii revealed an almost exclusive localization and accumulation of Pb within vascular bundles (Figure 2d). On the basis of the intensity of a Pb signal (Figure 2b), the concentration of Pb in the vascular bundles is likely to be many hundreds of times higher than that in the remainder of the stem. Image and intensity analysis revealed that 90.5% of the total Pb was restricted to the vascular bundles in the stem (Figure S5). Calcium was localized predominantly in nonvascular tissues (Figure 2c), while K and P were distributed

uniformly throughout the whole stem, with slightly higher accumulation in the vascular bundles and epidermis (Figure 2e,g). Zinc (Figure 2f) and S (Figure 2h) were both localized within the vascular tissues; however, statistical analysis demonstrated that only S intensity was positively correlated with Pb in these stem sections (correlation coefficient: r 2 ) 0.450, data number: n ) 123816, p < 0.01) (Figure S6c). To investigate in more detail the localization of elements in the vascular bundles, we performed µ-XRF focused on areas of greatest Pb signal strength (shown as rectangular areas in Figure 2d,f,h) by using a smaller step size (2 µm). The higher-resolution elemental maps of this area (Figure 3a) clearly show that the distribution patterns of Pb (red) and S (blue) are highly coincident, as indicated by the mixed (amaranth) color seen in Figure 3a, while Zn (pure green color) was more uniformly distributed through the tissue (Figure 3a). Statistical analyses also revealed a positive correlation between Pb and S localization within the vascular bundles (correlation coefficient: r2 ) 0.594, data number: n ) 12814, p < 0.001) (Figure 3b). No significantly positive VOL. 44, NO. 15, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Speciation of Pb. Bulk-EXAFS was employed to investigate the Pb speciation in the powdered fractions of the leaf and stem for both AE and NAE S. alfredii. This technique is able to provide basal information on Pb complexes in plant tissues. Figure S7 shows Pb LIII-edge EXAFS spectra of powdered plant samples (roots, leaves, and stems) of AE and NAE S. alfredii compared to model compounds, including Pb3(PO4)2, Pb(NO3)2, Pb(CH3COO)2, Pb-malate, Pb-GSH, Pb-citrate, and Pb-cell wall. The spectra were analyzed using linear combination fitting (LCF) (Table 1). An example of Linear Combination Fitting analyses of powdered stem sample Pb spectra with the standards was shown in Figure S8. This analysis revealed that the dominant (over 80%) form of Pb species in roots of both AE and NAE was similar to Pb-cell wall complex, suggesting absorbed aqueous Pb is converted into an organic Pb compound. The remaining proportion of the lead in the AE and NAE roots was comprised mainly of Pb3(PO4)2. Similarly, the stem and leaf samples of NAE also consist predominantly of the Pb-cell wall complex, representing 76.7% and 85.1%, respectively; however, in leaf and stem samples of AE, the percentage of the Pb-cell wall complex was lower. In comparison with the NAE, a small percentage of Pb in the tissues of AE S. alfredii appeared to be associated with SH-groups (GSH).

Discussion

FIGURE 3. Distribution patterns of Pb (red color), Zn (green color), and S (blue color) in the area shown in the boxes in Figure 2 (d, f, and h). The element of Zn is not collocated with the other two elements, indicated by pure green color. The elements of Pb and S collocated at a pixel spot, resulting in the mixed color indicated in the color mixing triangle. Beam size, 2*2 µm; step size, 2 µm. Scale bar: 20 µm. Correlation between XRF intensities of (b) Pb vs S and (c) Pb vs Zn were measured according to the XRF data. correlation between Zn and Pb was noted in this highintensity area (Figure 3c).

Metal compartmentalization in less bioactive cells such as epidermis has been hypothesized as one of the possible mechanisms for detoxification of heavy metals in plants and is thought to be particularly active in the hyperaccumulator and accumulator species (30). In previous studies in S. alfredii (26), the epidermis of leaf and stem was shown to be the main storage site for Zn. In the present study, with this same species, Pb was consistently restricted to vascular bundles of both leaf and stem, and very little Pb was accumulated in the parenchyma tissues outside the vascular tissues. The mobility of Pb in plants has been shown to be relatively low, and Pb is usually retained in the tracheid wall of the vascular bundles during transportation (31). The results shown here support the conclusion that there is a restriction of Pb transport from vascular tissue to the cortex and mesophyll cells. Micro-XRF not only allows for the nondestructive spatial visualization of metal abundance at the cellular level but also has the great advantage that it can be used to detect multiple elements simultaneously (26, 32, 33). Differential distribution patterns of various elements in the stem and leaf cross sections indicate different strategies involved in sequestration of these elements, while also suggesting the basal effectiveness of sample preparation methodology in this investigation. The presence of Pb complexed by thiol groups, probably phytochelatins (PCs), has been reported in other plants (23). However, previous studies indicated that no PCs were detected on the AE (named therein as “mined population”) S. alfredii, whereas a positive correlation between induced

TABLE 1. Lead Species Identified by Linear Combination Fit (LCF) Analyses of Powder Plant Samples Spectra Pb species (%) plant samples

NAE AE

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leaf stem root leaf stem root

Pb3(PO4)2

Pb-malate

Pb-GSH

Pb-cell wall

sum

residual (%)

16.4 ( 1.8 8.6 ( 2.2 18.5 ( 2.2 16.3 ( 2.3

16.4 ( 2.3 12.4 ( 1.2 27.8 ( 3.1 -

14.3 ( 2.5 17.1 ( 3.4 -

85.1 ( 4.1 76.7 ( 3.6 94.5 ( 2.5 70.7 ( 2.7 64.5 ( 4.4 87.6 ( 3.1

101.5 105.5 103.1 102.5 109.4 103.9

15.6 7.3 3.5 16.8 6.6 9.3

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GSH and Pb accumulation was observed (34). Recent research also indicated that the detoxification of Pb in Sedum alfredii is not related to PCs but the GSH (35) In this study, the similarities in the distribution patterns of Pb and S observed in tissues of AE plants indicated that Pb might be complexed by -SH compounds. However, the analysis of EXAFS on the plant tissues of two S. alfredii ecotypes revealed that only a small percentage of Pb in all tissues of AE S. alfredii appeared to be associated with SH-ligands (GSH), when compared with the NAE S. alfredii. The small proportion of Pb-GSH is hard to explain the 4-fold increase in Pb content in shoots of AE plants as compared with the NAE (Figure S2). Moreover, it should be noted here that the proportion of Pb-GSH in plant samples is less than 20%, and the LCF approach is not free of uncertainties and limitations (24, 28, 36). Although it is possible that the complex of Pb-GSH was damaged during analysis since the complex is sensitive to the beam, it is more likely that GSH complexation is not the main detoxification strategies in Pb tolerance in the AE plants. The most likely site for Pb compartmentation is the cell wall, and this is supported by the LCF analysis of EXAFS spectra. It is well-known that most of the Pb absorbed by plants is retained in the cell walls of the roots, possibly bound to pectin-like compounds, while some is translocated to the aerial parts of the plants. Kopittke et al. (22) suggested that the ability of signal grass Brachiaria decumbens Stapf to sequester insoluble Pb in the cell wall represents an important mechanism of Pb tolerance. Lead in the roots of Arabidopsis thaliana (L.) Heynh. also showed a good affinity to galacturonic acid, the main component of two pectin domains homogalacturonan and rhanogalacturoman, suggestive of the involvement of cell wall components in lead deactivation (37). In the present study, investigation of Pb species by EXAFS revealed that the dominant form of Pb species in roots, stems, and leaves of both ecotypes of S. alfredii was similar to the Pb-cell wall complex, suggesting a possible role of the cell wall component involved in Pb speciation in this plant species, not only in roots but also in the aerial parts of the S. alfredii plants. Results demonstrate that the mobility of Pb out of vascular tissues is very low in both ecotypes of S. alfredii and that Pb is largely retained in the cell walls, while SH-compounds may be involved in the Pb accumulation in AE S. alfredii.

Acknowledgments The work was supported by projects from the National Natural Science Foundation of China (30871589, 30630046, 20777068), and Key Project from Ministry of Environmental Protection of China (2011467057). This research was carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), USA (Proposal No.: 3186). Portions of this research were carried out at under approval of the Photon Factory Program Advisory Committee (Proposal No. 2006G134). The authors thank Dr. Atsuo Iida and Bart for data collection and Hongyun Peng, Dan Liu, Ejazul Islam for technical assistance.

Supporting Information Available Images of AE S. alfredii, Figure S1; biomass and Pb concentration, Figure S2; typical SRXRF microprobe spectra, Figure S3; relationships between Pb and other elements in AE leaf, Figure S4; percentages of Pb and other elements in different tissues of AE stem, Figure S5; relationships between Pb and other elements in AE stem, Figure S6; Pb LIII-edge EXAFS spectra, Figure S7; and linear combination fitting analyses of powdered stem sample with the standards, Figure S8. This material is available free of charge via the Internet at http://pubs.acs.org.

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