Characterization of a Lead Hyperaccumulator Shrub, Sesbania

Alabama State University, Montgomery, Alabama 36195. Lead phytoextraction can be economically feasible only when the developed systems employ high ...
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Environ. Sci. Technol. 2002, 36, 4676-4680

Characterization of a Lead Hyperaccumulator Shrub, Sesbania drummondii S H I V E N D R A V . S A H I , * ,† NATALIE L. BRYANT,† NILESH C. SHARMA,† AND SHREE R. SINGH‡ Biotechnology Center, Department of Biology, Western Kentucky University, 1 Big Red Way, Bowling Green, Kentucky 42101, and Department of Math and Science, Alabama State University, Montgomery, Alabama 36195

Lead phytoextraction can be economically feasible only when the developed systems employ high biomass plants that can accumulate greater than 1% Pb in their shoots. In this study Sesbania drummondii, a leguminous shrub occurring in the wild, was used to demonstrate its capability for greater than 1% Pb accumulation in shoots when grown in a Pb-contaminated nutrient solution. Shoot concentrations of >4% Pb were obtained from Sesbania plants grown on modified Hoagland’s solution containing 1 g Pb(NO3)2/L. The accumulation of Pb in the tissue was found to be dependent on the concentration of Pb in the nutrient solution. Addition of EDTA (100 µM) in the medium containing 1 g Pb(NO3)2/L increased uptake by 21%. Lower pH also favored Pb translocation to shoot. Results also indicate the path of Pb transport through root tissues. Scanning electron microscopy revealed the distribution of Pb granules in the cells from epidermis to the central axis, indicating both apoplastic and symplastic modes of transport. Transmission electron microscopy and X-ray microanalysis of root sections demonstrated the localization of Pb granules in the plasma membrane and cell wall, and also in the vacuoles. This investigation shows that S. drummondii satisfies the prerequisites for a hyperaccumulator, and thus might be useful, particularly, in the restoration of disturbed vegetation.

Introduction Lead concentration is increasing rapidly in the environment because of its increased use by human society. Alarming concentrations of the metal have been reported in water and land near industrial waste disposal sites. Lead (Pb) contamination originates mainly from cars and other petroleumfueled vehicles, metal smelting plants, mines, lead-contaminated sludges, and industrial wastes (1). Lead not only affects plant growth and productivity but also enters into the food chain causing health hazards to man and animals (2). Among the new strategies for removal of heavy metals from contaminated sites, phytoremediation is of growing interest because of its low environmental impact and costeffectiveness, even if a longer time is required for treatment * Corresponding author phone: (270) 745-6012; fax: (270) 7456856; e-mail: [email protected]. † Western Kentucky University. ‡ Alabama State University. 4676

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(3, 4). Many studies show that hyperaccumulators, plants that accumulate heavy metals from the soil into shoots, can be immensely useful in phytoremediation (5-8). The term hyperaccumulation was first used to describe plants containing >1000 mg/kg (0.1%) nickel in dry material (9). It now extends to represent a concentration of about 100 times greater than that of the highest values to be expected in nonaccumulating plants (10). Hyperaccumulators are usually small, native plants, such as those that belong to the genus Thlaspi and several others (11). Conversely, the plant in the present study, Sesbania drummondii, is a large bushy plant with high biomass production. In order for a plant species to be efficient for phytoextraction of metals, it should fulfill distinct prerequisites such as metal tolerance and metal accumulation via uptake-translocation (to aerial parts) and -sequestration. It should also have fast-growing biomass that is capable of high accumulation and easy harvest (12). The present investigation using a leguminous shrub has been designed to address questions with respect to this backdrop. Lead uptake and accumulation through roots of higher plants has been demonstrated in a variety of plant species (13-15). However, phytoextraction of lead from contaminated soils requires a better understanding of the mechanisms of lead uptake, translocation, and accumulation in plants (13). The present investigation aims to evaluate the capability of Sesbania drummondii for Pb uptake and accumulation from nutrient solution under conditions such as varying pH and use of a chelating agent. Use of aqueous environment for the measurement of Pb acquisition was preferred in this investigation as a model system before the trial is extended to soil at the greenhouse and field levels. This study also reveals the route of Pb transport and localization through cells and tissues of plant parts using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Feasibility of S. drummondii for phytoextraction of lead may be of immense value, particularly in restoration of disturbed vegetation near roadsides, abandoned mines, military sites, and dump and disposal sites, etc.

Experimental Section Germination of Sesbania drummondii Seedlings. Seeds were scarified in 85% sulfuric acid for 15 min and were washed under running tap water for 30 min. Seeds were then surfacesterilized with 0.2% mercuric chloride for 5 min, followed by three rinses with sterile distilled water. Nine surface-sterilized seeds were aseptically placed in each magenta vessel (97 mm high) containing 0.8% agar, and were allowed to germinate for 10 d at 25 °C under a light/dark regime of 16/8 h. Nutrient Solution and Treatments. Modified Hoagland’s medium (115 mg/L ammonium nitrate, 2.86 mg/L boric acid, 656 mg/L calcium nitrate, 0.08 mg/L cupric sulfate, 5.32 mg/L ferric tartrate, 240.7 mg/L magnesium chloride, 1.81 mg/L manganese chloride, 0.016 mg/L molybdenum trioxide, 300 mg/L potassium nitrate, and 0.22 mg/L zinc sulfate) was used as a basal nutrient medium for the growth of the seedlings. The basal medium was supplemented with Pb(NO3)2 (Sigma, analytical grade) at the rate of 0-1500 mg/L. The pH of the medium was adjusted to 5.8 with 1 M HCl. The values given in this report correspond to the mean (( S.E.) of 3-6 replicates. EDTA Treatment. Disodium salt of ethylenediaminetetraacetic acid (0-500 µM) was added in the nutrient solution containing 1000 mg/L Pb(NO3)2, and pH was adjusted to 6.8 to allow maximum dissolution of EDTA. 10.1021/es020675x CCC: $22.00

 2002 American Chemical Society Published on Web 09/25/2002

pH Variation. To determine the effect of pH on uptake of Pb by Sesbania, the pH of the solution was varied from 3.7 to 7.7 at the fixed concentration of Pb(NO3)2 (1000 mg/L). Transfer of Seedlings and Incubation. Individual seedlings (8-10 cm) were aseptically transferred to a culture tube (15 × 2.5 cm) containing 5 mL of nutrient solution with or without Pb(NO3)2 in the presence or absence of EDTA. Cultures were incubated at 22 ( 2 °C under 16/8 h of light/ dark regime for 2 weeks. Observations for 3-6 replicates were recorded in each treatment. To confirm whether Pb transport to different tissues is irreversible, seedlings grown with Pb(NO3)2 for 2 weeks were taken out, washed, and recultured in basal nutrient medium [in absence of Pb(NO3)2]. They were grown for another week in the condition as described above. Pb Analysis of Treated Seedlings. The harvested seedlings were rinsed three times with distilled water, separated into roots and shoots, and oven-dried. Samples were weighed and placed in a 15-mL screw capped Teflon beaker. Concentrated HNO3 (3 mL) was added to the sample, and the beaker was placed on a hot plate at a temperature of 100 °C overnight, and the contents were then evaporated to dryness. Samples were allowed to cool and made up gravimetrically with 2% HNO3 to a volume of 20 mL. The ICP-OES analysis was carried out using external calibration procedure, and Y (0.1 ppm) was used as an internal standard to correct for drift and matrix effect (16). Statistical Analysis. Statistical analysis was carried out using SYSTAT (Version 9 for Windows, 1999, Systat Software Inc., Richmond, CA). Pearson correlation analysis was performed with Bonferroni corrections. Scanning Electron Microscopy. The harvested seedlings were washed thoroughly with distilled water and quick-frozen by plunging them into liquid nitrogen slush. Seedlings were then transferred to liquid nitrogen where they were fractured into small pieces. The pieces were placed in Styrofoam cups, each containing liquid nitrogen and a 500-g steel block. The pieces, such as secondary root, lower stem, upper stem, and leaf, were freeze-dried by warming them to room temperature in a bell jar evacuated with a rotary pump to 20 milliTorr. Dried Sesbania pieces were mounted on all stubs using double-sided carbon tape. Samples were viewed uncoated in a JEOL 5400 LV SEM at 15 kV low vacuum mode using a backscattered electron detector. Element analysis was carried out using an attached KEVEX Sigma energy dispersive X-ray spectrometer (EDX), otherwise called X-ray microanalysis. Transmission Electron Microscopy. After the samples were washed, pieces of the roots were cut at room temperature in 2% glutaraldehyde in 50 mM PIPES (pH 6.8). These pieces were briefly placed in a vacuum to aid infiltration of the fixative into the tissues and incubated in the fixative for 2 h at room temperature. The samples were then washed in 50 mM PIPES buffer, and post-fixed in 2% OsO4 in water for 2 h at room temperature. After the samples were washed in water, they were dehydrated through an ethanol series and embedded in Spurr’s resin. Thin sections were prepared by a microtome (RMC, MT-X), collected on Cu-support grids, and observed with a JEOL 120 CX TEM at 80 kV. Element analysis was carried out at 40 kV using IXRF Systems energy dispersive X-ray spectrometer (EDX), otherwise called X-ray microanalysis.

Results and Discussion Sesbania drummondii seedlings tolerated high levels (2501500 mg/L) of Pb(NO3)2 when grown in modified Hoagland’s nutrient medium under sterile conditions. However, seedlings showed symptoms of some toxicity (in the form of stunted growth) at and above 1000 mg/L Pb(NO3)2. On the basis of the concentration of Pb(NO3)2 in the medium, plant

FIGURE 1. Accumulation of Pb in Sesbania shoot grown on modified Hoagland’s medium containing 0-1500 mg/L Pb(NO3)2. Values represent mean (( SE) of 6 replicates. parts accumulated Pb in their tissues. Lead accumulation in shoots was positively correlated with Pb concentrations in the medium (r ) 0.8). The relationship between Pb accumulation and medium Pb concentrations was statistically significant based on Bonferroni-adjusted probability (P ) 0.026). The optimal concentration of medium Pb for accumulation was 1000 mg/L, which Sesbania could tolerate. Shoots accumulated >4% of Pb in their dry weight at 1000 mg/L Pb(NO3)2 in 2 weeks (Figure 1). The amount of Pb uptake by roots reached 60 g/kg (6%) dry weight after one week of treatment with 500 mg/L Pb(NO3)2 (Figure 2). Root accumulation of Pb was positively correlated with time (days) (r ) 0.874). Accumulation was significantly different at 1 and 3 days (P ) 0.0001). The amount of Pb concentrated by aerial parts in this species is far greater than that of the known highest Pb-accumulator, Indian mustard, which accumulates 1.5% Pb in shoot tissues when grown in nutrient solution with high concentration of soluble Pb (17). At lower Pb concentrations in solution, accumulations in Sesbania shoots were significantly less (P < 0.001) than the shoot accumulations at higher exogenous Pb concentrations, although root Pb concentrations were higher (as much as 6% of dry weight) at those concentrations. This trend is comparable to that reported for Indian mustard (17). Shoot Pb observed in this study is much higher than that of other high accumulators, Thlaspi rotundifolium (18) and Pelargonium species (15), for which shoot accumulations were 8.5 and 3.0 g/kg dry weight, respectively. Sesbania species also compares with corn (13) and scented geranium (15) in its potential for tolerance to high levels of soluble Pb (up to 1500 mg/L). Several chelating agents such as EDTA, CDTA (trans-1, 2 cyclohexylenedinitrilotetra acetic acid), DTPA (diethylenetrinitrilotetraacetic acid), and citric acid have been used for fortification of Pb-contaminated soils in order to increase the lead bioavailability and uptake (13, 14, 19). Among these chelating agents, EDTA was shown to be the most efficient. It enhanced shoot Pb concentrations by 120-fold in corn growing in soils containing 2500 mg/kg Pb (13), and 150-fold VOL. 36, NO. 21, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Lead accumulation by Sesbania root grown on modified Hoagland’s medium containing 500 mg/L Pb(NO3)2 in 7 days. Values represent mean (( SE) of 6 replicates.

FIGURE 3. Accumulation of Pb by Sesbania shoot grown in modified Hoagland’s medium containing 1000 mg/L Pb(NO3)2 and 0-500 µM EDTA. Values represent mean (( SE) of 5 replicates. in Indian mustard grown in soils contaminated with 600 mg/ kg Pb (14). However, this study demonstrates only a marginal increase of 21% Pb in Sesbania shoot when 100 µM EDTA was applied to the nutrient solution containing 1000 mg/L Pb(NO3)2 (Figure 3). The physiological basis of uptake of PbEDTA complex, and particularly the migration of this negatively charged large molecule across the membranes, is unknown (20), but earlier results indicate that EDTA and other chelating agents enhance Pb transport into the xylem, and also Pb translocation from roots to shoots and aerial 4678

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FIGURE 4. Effect of pH on accumulation of Pb in Sesbania shoot grown in modified Hoagland’s medium supplemented with 1000 mg/L Pb(NO3)2. Values represent mean (( SE) of 3 replicates. parts (13, 14). Wu et al. (21) have reported that EDTA appears to increase plant transpiration, which is the driving force in phytoextraction of the Pb-chelate complex from soil. The low impact of EDTA on Pb accumulation in Sesbania can be correlated with the fact that plants in this experiment were grown in sterile solution in test tubes fitted with closures. Therefore, transpirational pull being the least, EDTA could not influence the rate of uptake greatly. Soil pH controls Pb solubility by influencing its bioavailability to crops. The uptake of Pb from hydroponic solutions was also strongly related to solution pH in the presence or absence of EDTA (14). To determine whether pH variation affects the rate of Pb accumulation in Sesbania shoot, seedlings were grown in lead-contaminated nutrient solution under varying pH. The results demonstrated an increase in uptake with decreasing pH (Figure 4). However, the increase was only 16% at pH 3.7 over values at pH 5.8. These results are in agreement with those of Indian mustard, in which EDTA-facilitated uptake in shoots increased 3-fold the values obtained without EDTA at pH 3.5 (14). Probably, the efficacy of EDTA on uptake kinetics of Sesbania would have been different under lower pH. To understand the metal detoxification mechanism(s) in Sesbania drummondii, it is necessary to map the pathway of Pb transport and sites of Pb localization. Thus, plant parts (root, lower stem, upper stem, and leaf) treated with 1000 mg/L Pb(NO3)2 were examined by SEM and TEM. The SEM of root tissue revealed bright spots of Pb, forming patterns of concentric circles inside the stele, more precisely in the central region of vascular bundles. These spots were also visible on the surface of root epidermis, in the parenchyma cells of the cortical region and outside the endodermis (Figure 5A). The basal part of the stem also revealed Pb deposits in the same fashion, but they were more like clumps or sheets (Figure 5C). Lead deposits were also observed in upper stem and leaf regions, more or less in the same fashion, but in low amounts. These deposits in the upper stem appeared to be sitting on the fractured faces of the cells. Control root and stem sections did not show such deposits or spots anywhere in SEM (Figure 5B,D). X-ray microanalysis of these deposits confirmed them as Pb granules (Figure 5E), which were not

FIGURE 5. Scanning electron micrographs of root and stem of Sesbania drummondii seedlings grown in modified Hoagland’s medium containing 1000 mg/L Pb(NO3)2. (A) Root section (scale marker represents 100 µm) shows dense Pb particles (arrowheads) distributed from epidermis through central cylinder region. (B) Control root section (scale marker represents 100 µm) showing absence of Pb deposits. (C) Basal stem section (scale marker represents 250 µm) shows dense regions of Pb deposition (arrowheads) from epidermis through central cylinder. (D) Control stem section (scale marker represents 250 µm) showing absence of Pb deposition. (E) X-ray microanalysis spectrum of one of the root regions shown in A. (F) X-ray microanalysis spectrum of control root shown in B. observed in the control (Figure 5F). Microanalysis spectra through certain points on the root section demonstrated a decreasing gradient of Pb contents from epidermis to the central axis. These observations provide clues for both active and passive mechanisms of Pb uptake through Sesbania roots. Both apoplastic migration and symplastic transportation of Pb have been shown to occur in Pelargonium species (15) and onion root (22). In another study, plants treated with 1000 mg/L of Pb(NO3)2 for two weeks were transferred to nutrient medium (in absence of Pb) for a week in order to assess whether absorbed Pb leaches back to the solution on transfer to the lead-free medium. The presence of Pb in stem and leaf in the same fashion under SEM confirms the irreversible nature of uptake and translocation. Cellular and sub-cellular localization of Pb in root cells was evaluated using TEM and EDX. In all the cell types of the root, Pb deposits were found along the plasma membrane of the cells. The smaller deposits appeared to coat the surface of the plasma membrane, while large deposits looking like globules extended deeper into the cell wall, sometimes pushed up against the plasma membrane (Figure 6A,B). Some large deposits were observed in vacuoles, partially

separated from the cell membrane deposits by a layer of cytoplasm. X-ray spectra of these deposits demonstrated peaks of Pb, O, and P (Figure 6C). The control root showed no evidence of cellular or subcellular Pb deposits. The precise mechanism of detoxification in Pb-tolerant plants is yet to be known. The TEM-EDX and extended X-ray absorption fine structure (EXAFS) studies have established that cell walls play an important role in the accumulation of Pb in heavy-metal-tolerant grass Agrostis capillaries (23). This species presents extra cellular Pb-containing grains in the outermost layer of root cells; the grains are predominantly composed of pyromorphite, a lead phosphate mineral. Sarret et al. (20), using TEM-EDX, has also shown in Phaseolus vulgaris leaf that the metal accumulates near the cell membrane and in chloroplasts. The TEM-EDX examination of Sesbania roots confirms Pb deposition in cell membrane and wall, but it also demonstrates another site of intracellular localization, i.e., vacuoles (Figure 6B). This way, Sesbania might be protecting itself from the toxic effects by dumping Pb into its vacuoles. The TEM-EDX spectra of cell deposits also showed peaks of P and O, leading to the belief that Pb is sequestered as lead phosphate. Another explanation for high accumulation of the metal in this plant may be due to VOL. 36, NO. 21, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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gratefully acknowledge the valuable assistance of Dr. J. Andersland with electron microscopy. The assistance of S. Cheepala in statistical analysis is duly acknowledged.

Literature Cited

FIGURE 6. Transmission electron micrographs of root cells of Sesbania drummondii grown in modified Hoagland’s medium containing Pb. (A) Three cortical cells bordering a triangular intercellular space, show Pb deposits (indicated with arrowheads; scale marker represents 2 µm) on their plasma membranes. (B) Magnified view (scale marker represents 0.2 µm) of one of the Pb aggregates in A. A part of the deposit (arrowhead) is outside the plasma membrane (PM), in the cell wall (CW), while another part is inside the tonoplast (T) in the vacuole (V). (C) X-ray microanalysis spectrum of a Pb deposit shown in A. a greater number of functional groups present on its cell membrane/wall that bind with metal (24). The phytochelatins, sulfur-containing proteins, have been advocated to complex metals in plants, but this hypothesis of metal detoxification has not yet been demonstrated for lead (20).

Acknowledgments This work was supported in part through Kentucky NSFEPSCoR grant (4-62905-99-218) awarded to S.V.S. We

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Received for review April 4, 2002. Revised manuscript received August 8, 2002. Accepted August 12, 2002. ES020675X