Environ. Sci. Technol. 2008, 42, 4595–4599
Localization and Chemical Speciation of Pb in Roots of Signal Grass (Brachiaria decumbens) and Rhodes Grass (Chloris gayana) P E T E R M . K O P I T T K E , * ,†,‡ COLIN J. ASHER,† F. PAX C. BLAMEY,† GRAEME J. AUCHTERLONIE,§ YANAN N. GUO,| AND N E A L W . M E N Z I E S †,‡ School of Land, Crop and Food Sciences, The University of Queensland, St. Lucia, Queensland, Australia, 4072, Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC-CARE), The University of Queensland, St. Lucia, Queensland, Australia, 4072, Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia, Queensland, Australia, 4072, and School of Engineering, The University of Queensland, St. Lucia, Queensland, Australia, 4072
Received October 18, 2007. Revised manuscript received March 15, 2008. Accepted April 3, 2008.
Lead (Pb) contamination of soils is of global importance but little is known regarding Pb uptake, localization, or the chemical forms in which Pb is found within plants, or indeed how some plants tolerate elevated Pb in the environment. Two grasses, signal grass (Brachiaria decumbens Stapf) (Pbresistant) and Rhodes grass (Chloris gayana Kunth) (Pb-sensitive), were grown for 14 d in dilute nutrient solutions before examination of roots using transmission electron microscopy (TEM) to determine the distribution and speciation of Pb in situ. In both grasses, Pb was initially present primarily in the cytoplasm of rhizodermal and cortical cells before being sequestered within vacuoles as the highly insoluble (and presumably nontoxic) chloropyromorphite (Pb5(PO4)3Cl). In signal grass, Pb also accumulated within membranous structures (perhaps the Golgi apparatus), prior to apoplastic sequestration as chloropyromorphite. These findings suggest that the ability of signal grass to sequester insoluble Pb in the cell wall represents an additional and potentially important mechanism of Pb tolerance not possessed by the Pb-sensitive Rhodes grass.
Introduction Lead is a significant environmental pollutant, being released from a variety of sources including mining, transport, and smelting of Pb-ores, metal manufacturing, and the burning of leaded fuels. However, little is known about (i) the chemical species (forms) that accumulate in plants exposed to Pb, (ii) * Corresponding author e-mail:
[email protected]; phone: +61 7 3346 9149; fax: +61 7 3365 1177. † School of Land, Crop and Food Sciences. ‡ Cooperative Research Centre for Contamination Assessment and Remediation of the Environment. § Centre for Microscopy and Microanalysis. | School of Engineering. 10.1021/es702627c CCC: $40.75
Published on Web 05/17/2008
2008 American Chemical Society
the location where Pb accumulates, and (ii) the mechanisms which allow some plants to resist Pb. Although the identification of Pb-speciation within plants is crucial to understanding how plants tolerate Pb, there is no conclusive evidence as to the forms of Pb on a cellular or subcellular level. For example, Cotter-Howells et al. (1) reported that Pb accumulates in the roots of soil-grown Agrostis capillaris L. (brown-top bent grass) as chloropyromorphite (Pb5(PO4)3Cl), but this was only identified after the roots had been ashed at 400 °C for 8 h. Therefore, the original subcellular location of these Pb-deposits is not known and it is unclear whether the ashing process modified the speciation of the Pb. Shoots of Phaseolus vulgaris L. (bean) accumulate PbCO3 (2) but the subcellular location of these deposits was not determined. Synchrotron-based X-ray absorption spectroscopy (XAS) studies (3) have demonstrated that Pb accumulates as Pb-acetates and Pb-sulfides in the roots of Sesbania drummondii (rattlebush); however, once again, the cellular and subcellular location of this Pb was not determined. Furthermore, these results apparently conflict with the transmission electron microscopy (TEM) energy dispersive X-ray spectroscopy (EDS) data of the same study (3) that showed the Pb-deposits were associated with P. Also investigating Sesbania drummondii, Sahi et al. (4) reported that Pb in the roots was present as an unidentified Pbphosphate. Many solution culture studies investigating Pb phytotoxicity have used Pb concentrations several orders of magnitude greater than those found in the soil solution of contaminated sites; for example, up to 1700 µM (2) and 2400 µM Pb (3) c.f. those reported for soil solutions at contaminated sites, viz. e 15 µM (5) and e1.1 µM Pb (6). The use of unrealistically high Pb concentrations in solution raises questions about the value of such studies. The use of dilute nutrient solutions as found in soil solutions has shown (7) that signal grass (Brachiaria decumbens Stapf) is much more resistant than Rhodes grass (Chloris gayana Kunth) to Pb in the root environment, but the study was not able to determine the reason for the difference between the species in Pb sensitivity. The objectives of the current work, therefore, were to use (i) TEM to examine the distribution of Pb within roots and (ii) TEMbased analytical techniques to determine the chemical composition of the Pb-deposits in Pb-intoxicated roots of the two perennial grass species
Materials and Methods Plant Growth. Seedlings of signal grass (cv. Basilisk) and Rhodes grass (cv. Pioneer) were transplanted into 22 L of nutrient solutions at pH 4.75 (7). There were four treatments: 0 and 20 µM Pb for signal grass and 0 and 5.5 µM Pb for Rhodes grass, concentrations which decrease root mass of these species by ca. 85% (7). Two replicates (each of four plants) were established for each treatment, and the plants were grown for 14 d. Nutrients were periodically added to the solution to replace those removed by the plants. The solutions were sampled after 0, 6, 10, and 14 d, filtered (0.22 µm Millipore GSWP), acidified to pH < 2.0 using 20 µL of concentrated HCl, and refrigerated (3.5 °C) before analysis by inductively coupled plasma mass spectrometry (ICPMS) for Pb. The measured mean Pb concentrations were 0 and 15 µM for signal grass, resulting in calculated activities of 0 and 10 µM Pb2+ using PhreeqcI 2.11 with the Minteq database (8). Corresponding measured values for Rhodes grass were 0 and 5.1 µM Pb, with calculated activities of 0 and 3.4 µM Pb2+. VOL. 42, NO. 12, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Microscopy. After 14 d growth in the nutrient solutions, the root tips (0-15 mm) were harvested, immediately placed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, and stored at 3.5 °C. The samples were postfixed with 1% osmium tetroxide (OsO4, a stain for lipids in membranes) (9). Ultrathin (ca. 60-90 nm) sections were then cut on a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and digital images were captured using a JEOL 1010 TEM (JEOL Ltd., Tokyo, Japan) at either 80 or 100 kV. It was assumed that all electron-dense (i.e., dark-colored) deposits were Pb, with this later being confirmed by EDS. No electron-dense material was observed in the sections taken from the roots of control (0 µM Pb) plants. Electron-dense deposits were analyzed by EDS using a Philips Tecnai F20 (FEI Company, Oregon) field emission gun (FEG) analytical electron microscope (AEM) at 80 kV in scanning transmission electron microscopy (STEM) mode with an EDAX (EDAX, New Jersey) thin-window EDS detector. Calculations for quantitative EDS analysis were performed offline using ES Vision v4.0.172 (FEI Company). The ultrathin sections were also analyzed by selected area electron diffraction (SAED) (Philips Tecnai F20 FEG-AEM at 200 kV). However, SAED damaged the Pb-deposits, and the 1-5% error of electron diffraction prevented the generation of useful results. The EDS analyses for elemental composition of root tissue suggested two possible forms in which Pb might occur, viz. chloropyromorphite (Pb5(PO4)3Cl) and lead phosphate (Pb3(PO4)2). The former was prepared by reacting 250 mL of 0.5 M Pb(NO3)2 with 250 mL of 0.3 M HNa2PO4 and 0.1 M NaCl that X-ray diffraction (XRD) studies (1) had confirmed to form chloropyromorphite. A standard of Pb3(PO4)2 was prepared by reacting 250 mL of 0.5 M Pb(NO3)2 with 250 mL of 0.33 M HNa2PO4. Both standards were placed on holey carbon-coated copper grids for EDS analysis. To complement the results obtained by EDS, electron energy loss spectroscopy (EELS) (Philips Tecnai F30 G2 FEGAEM at 300 kV) was also used to examine the electron-dense deposits. In this instance, roots were high-pressure frozen in liquid nitrogen, freeze substituted at -85 °C in acetone, and warmed to -50 °C before being embedded in Lowicryl HM20 resin.
Results Cellular and Subcellular Distribution of Pb. Examination of the signal grass roots using TEM revealed that, at 1 mm from the apex, all of the Pb accumulated within the rhizodermis and outer-cortex, with no Pb-deposits observed in the inner-cortex or stele. The Pb in the rhizodermis (not presented) and outer-cortex was not obviously crystalline (Figure 1). These Pb-deposits tended to be within the cytoplasm, with some present in membrane-bounded vesicles and vacuoles (Figure 1). It was evident from these and other images that the Pb which first accumulated within the cytoplasm was gathered into membrane-bounded vesicles and subsequently sequestered within the vacuole, apparently by exocytosis (Figure 1c and d). Comparatively few deposits were present in the cell wall and intercellular spaces (Figure 1b). At a distance of 15 mm from the signal grass root apex (i.e., in older root tissue), the Pb was distributed more widely throughout the cortex, with substantial accumulation near the endodermis (Figure 2a). Some vacuolar Pb deposits were observed throughout the rhizodermis and outer cortex (Figure 2b and c), but much of the Pb was apoplastic (Figure 2a, d, h). These deposits were almost exclusively crystalline, forming long acicular (needle-like) crystals (Figure 2d and h). High magnification examination of transverse sections of the larger crystals revealed their hexagonal cross-section (Figure 2e). Cell walls surrounding these crystalline Pb deposits were 4596
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FIGURE 1. Transmission electron micrographs of outer cortical cells of sections taken either 1 or 15 mm from the apex of roots of signal grass (Pb2+ activity of 10 µM) or Rhodes grass (Pb2+ activity of 3.4 µM). C ) cytoplasm, V ) vacuole, CW ) cell wall, I ) intercellular space, MV ) membrane-bounded vesicle. abnormally thick (often up to 5 µm) compared to typical values of ca. 0.3-1 µm (Figure 2d and h). Strand-like deposits were observed in the cytoplasm and vacuole in some of the cortical cells (Figure 2f and Figure 3), and these were often present close to the acicular deposits (Figure 2h). Remnants of these strand-like deposits were evident in what appeared to be recently formed apoplastic deposits that were identified by increasing the image brightness (i.e., increasing the electron beam intensity) (Figure 3a and b). Finally, many of the strand-like deposits were similar in shape to the acicular deposits in the cell walls (Figure 3a/c, d/e, f/g, h/i). As with signal grass, all of the Pb deposits observed 1 mm from the Rhodes grass roots apex were located within the rhizodermis and outer-cortex, with no Pb evident in the innercortex or stele. These Pb deposits generally appeared to lack a clearly visible crystal structure although some small acicular crystals were present. These deposits were predominantly intracellular (Figure 1e), with few Pb deposits in the apoplast (Figure 1f). At a distance of 15 mm from the root apex, some Pb was observed within the apoplast (Figure 1g), but most of the Pb was sequestered in large vacuoles (Figure 1h). It is noteworthy that no electron-dense deposits were found in the stele of signal grass or Rhodes grass roots, even in older tissue. Composition of Pb Deposits. All electron-dense deposits in root tissue analyzed by EDS showed peaks for O, Cu, Pb, and Cl (Figure 4c, e, f, g), and all but one (Figure 4d) showed peaks for P. In this instance, any peak for P would have been
FIGURE 2. Transmission electron micrographs of sections 15 mm from the apex of signal grass roots after 14 d growth in dilute nutrient solutions at a Pb2+ activity of 10 µM. C ) cytoplasm, V ) vacuole, CW ) cell wall, I ) intercellular space. Note the hexagonal crystals in (e) (in particular, see arrow). Note that (c) is a close-up of the large vacuolar Pb-deposits (outer cortex) to the right in (b), and that (g) is a close-up of the strand-like deposit (inner cortex) below the horizontal strands in (f). obscured by the strong Os signal (Os indicating the presence of membranous lipids). Most spectra also contained a peak for C (possibly from the surrounding plant tissue) and some small Ca peaks were observed in some spectra (Figure 4c and g). The presence of Cu in the analyses is attributed to the use of Cu grids for holding the sample, and Si (Figure 4e and f) is from the use of a glass knife. Overall, the spectra for the electron-dense deposits were qualitatively similar to that for the chloropyromorphite standard (Figure 4b). The large size of the apoplastic Pb deposits (up to several micrometers) aided in their analysis. These large deposits consisted of numerous, small hexagonal crystals (Figure 2e) which may be indicative of a number of compounds. A search was made of the Inorganic Crystal Structure Database (10) to reveal compounds which form hexagonal crystals and which are consistent with the elemental composition revealed by the EDS analyses. Three possible compounds were identified: Pb5(PO4)3Cl, Pb5(PO4)3OH, and Pb3(PO4)2. Clear speciation of these compounds by EDS is difficult because (i) all three compounds have a similar Pb:P:O ratio and (ii) the Cl KR peak and the Pb Mγ peaks overlap (Figure 4a and b). To overcome these problems, 10 EDS spectra of each standard and Pb-deposit were collected, averaged, and scaled relative to the Pb MR1 peak (Figures 4 and 5). The apoplastic Pb-deposit curve in the region of interest was found to more closely match the curve of the Pb5(PO4)3Cl (chloropyromorphite) than that of Pb3(PO4)2 (Figure 5a). Further, quantitative calculations of the EDS spectra from the apoplastic deposits indicated that the average atomic ratio of Pb:P:O:Cl was 5.0: 3.4:11.8:0.98, a result in agreement with the ratio of 5.0:3.0:
FIGURE 3. Transmission electron micrographs of cortical cells 15 mm from the apex of signal grass roots after 14 d growth in dilute nutrient solutions at a Pb2+ activity of 10 µM. C ) cytoplasm, V ) vacuole, CW ) cell wall, I ) intercellular space. Note that (b) is of the same deposit as in (a) except at higher magnification and electron beam intensity. 12:1.0 for Pb5(PO4)3Cl. Subsequently, the superior resolution of EELS confirmed the presence Pb, P, O, and Cl as expected for Pb5(PO4)3Cl (data not presented). The small size (often 10 mM (22, 23). Acicular crystals have been reported in Pb studies with cowpea (Vigna unguiculata L.) (9), carthusian pink (13), sweet vernal grass (Anthoxanthum odoratum L. Walp.) (24), and maize (12). It is possible, therefore, that chloropyromorphite was present in tissues of these species, encouraging further research to determine the overall importance of chloropyromorphite in Pb tolerance. Studies with sweet vernal grass (24), radish (Raphanus sativus L.) (25), maize (26), and sago pondweed (Potamogeton pectinatus L.) (27) have shown that Pb accumulates predominantly within the apoplast. This was evident also in the present study with tissue sampled 15 mm from the root apex of the more tolerant signal grass. Importantly, the present study has also shown that initially (i.e., within 1 mm of the root apex), almost all of the Pb accumulated intracellularly in both grasses, with only minor traces of Pb in the apoplast (Figure 1). In neither species was Pb evident in the stele. We consider that the accumulation of Pb in membranous structures and the subsequent movement into the apoplast (where it is stored as chloropyromorphite) by signal grass contributes to its higher Pb tolerance compared to Rhodes grass. Further investigations are required to establish the mechanisms by which Pb2+ is toxic to plant metabolism, and to determine which plant species employ apoplastic sequestration of chloropyromorphite as does signal grass to mitigate toxic Pb effects.
Acknowledgments Rick Webb, Robyn Webb, and Professor John Drennan from the Centre for Microscopy and Microanalysis at The University of Queensland are acknowledged. This research was funded through the Cooperative Research Centre for Contamination, Assessment and Remediation of the Environment (CRC-CARE) Project 3-3-01-05/6.
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