Sorghum Roots are Inefficient in Uptake of EDTA-chelated Lead
1 Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan
2 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
* For correspondence. E-mail: maj{at}rib.okayama-u.ac.jp
Received: 24 October 2006 Returned for revision: 9 January 2007 Accepted: 30 January 2007
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ABSTRACT |
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Background and Aims: Ethylene diamine tetraacetic acid (EDTA)-assisted phytoremediation has been developed to clean up lead (Pb)-contaminated soil; however, the mechanism responsible for the uptake of EDTA–Pb complex is not well understood. In this study, the accumulation process of Pb from EDTA–Pb is characterized in comparison to ionic Pb [Pb(NO3)2] in sorghum (Sorghum bicolor).
Methods: Sorghum seedlings were exposed to a 0·5 mM CaCl2 (pH 5·0) solution containing 0, 1 mM Pb(NO3)2 or EDTA–Pb complexes at a molar ratio of 1:0·5, 1:1, 1:2 and 1:4 (Pb:EDTA). The root elongation of sorghum at different ratios of Pb:EDTA was measured. Xylem sap was collected after the stem was severed at different times. The concentration of Pb in the shoots and roots were determined by an atomic absorption spectrometer. In addition, the roots were stained with Fluostain I for observation of the root structure.
Key Results: Lead accumulation in the shoots of the plants exposed to EDTA–Pb at 1:1 ratio was only one-fifth of that exposed to ionic Pb at the same concentration. Lead accumulation decreased when transpiration was suppressed. The concentration of Pb in the xylem sap from the EDTA–Pb-treated plants was about 1/25 000 of that in the external solution. Root elongation was severely inhibited by ionic Pb, but not by EDTA–Pb at a 1:1 ratio. Root staining showed that a physiological barrier was damaged in the roots exposed to ionic Pb, but not in the roots exposed to EDTA–Pb.
Conclusions: All these results suggest that sorghum roots are inefficient in uptake of EDTA-chelated Pb and that enhanced Pb accumulation from ionic Pb was attributed to the damaged structure of the roots.
Key words: Complex, EDTA, form, Pb, phytoremediation, uptake system, sorghum (Sorghum bicolor)
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INTRODUCTION |
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Since the dawn of the Industrial Revolution, heavy metals have been used widely by mankind and have accumulated in the environment at an exponential rate (Nriagu, 1996). Lead (Pb) is one of the most common toxic metals and exhibits specific toxicity, especially for young children (Mushak, 1993). Soils are a major and terminal sink for lead deposition in the environment. Unlike organic contaminants, Pb cannot be degraded once the soil has been contaminated by this element. Various soil treatments may be employed to reduce the in situ bioavailability of Pb to plants and animals (e.g. Brown et al., 2004); however, for long-term sustainability it is preferable to remove Pb from contaminated soils.
Phytoremediation has been proposed as an environmentally friendly and cost-effective plant-based technology for cleaning up contaminated soil (Salt et al., 1998; McGrath et al., 2002). However, there are two limitations for the phytoremediation of Pb. First, Pb has an extremely low solubility in soils due to complexation with organic matter, sorption on oxides and clays, and precipitation as carbonates, hydroxides and phosphates (McBride, 1994). Low availability of Pb limits its uptake by plants. Second, there are few natural hyperaccumulators of Pb for use in phytoextraction.
To increase Pb availability in soils, some synthetic chelates have been used and EDTA is among the most effective to solubilize Pb in soil and to enhance plant uptake (Blaylock et al., 1997; Huang et al., 1997; Wu et al., 1999). However, inconsistency in the literature exists on the effect of chelates on metal uptake or accumulation. Some authors reported that EDTA application did not enhance or even decrease heavy metal uptake (Athalye et al., 1995), while others observed a significant increase after EDTA application (Blaylock et al., 1997; Huang et al., 1997; Vassil et al., 1998). This inconsistency may be attributed to many factors including different plant species, experimental conditions, concentrations, and ratio of EDTA to Pb. In the present study, chelated Pb (EDTA–Pb) was compared with ionic Pb for their effects on Pb accumulation, Pb toxicity and root structure in sorghum. The results show that the plant roots of sorghum are inefficient in uptake of EDTA–Pb complex.
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MATERIALS AND METHODS |
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Plant materials and growth conditions
Seeds of sorghum (Sorghum bicolor L. ‘Early Sumac’) were rinsed in deionized water and transferred to a Petri dish with moist filter paper overnight at 25 °C. Germinated seeds were placed on nets floating on a solution containing 0·5 mM CaCl2 (pH 5·0) in 1·5-L plastic containers and incubated for 4 d. The solution was renewed daily. Six-day-old seedlings of similar size were selected and pre-cultured in an aerated nutrient solution containing one-fifth-strength Hoagland solution (pH 5·6). The nutrient solution contained the following macronutrients (mM): KNO3 (1), Ca(NO3)2 (1), MgSO4 (0·4), and (NH4)H2PO4 (0·2); and micronutrients (µM): Fe-EDTA (20), H3BO3 (3), MnCl2 (0·5), CuSO4 (0·2), ZnSO4 (10) and (NH4)6Mo7O24 (1). After 15–21 d of culture, the seedlings were used for the following experiments. All experiments described below were conducted in a growth chamber with 16 h light and 8 h dark at 25 °C. Each experiment was repeated at least twice, with each treatment being replicated three times in an experiment.
Pb uptake from different chemical forms
Seedlings, as prepared above, were exposed to a 0·5 mM CaCl2 (pH 5·0) solution in 50-mL plastic bottles containing 1 mM Pb and different concentrations of Na2EDTA, resulting in different molar ratios (Pb:EDTA) of 1:0·5, 1:1, 1:2 and 1:4. Two days later, shoots and roots were harvested separately and rinsed three times with a 0·5 mM CaCl2 solution to avoid contamination by the culture solution. The samples were oven dried at 70 °C for 3 d and then their dry weights were recorded. Dried plant material was ground and digested in concentrated nitrate acid. Concentration of Pb was determined using the method described below.
A time-course experiment was performed by transferring the seedlings to a 0·5 mM CaCl2 solution (pH 5·0) containing Pb(NO3)2 and EDTA–Pb (1:1) complex at 1 mM. At 0, 3, 6, 9, 12 and 24 h, shoots and roots were sampled as described above.
Xylem sap collection
Seedlings (21-d-old) were exposed to a solution containing 1 mM Pb(NO3)2 or 1 mM EDTA–Pb (1:1) complex at pH 5·0. At 0, 0·5, 1, 2, 4 and 8 h after exposure, the stems were severed at 1 cm above the roots, and the xylem sap was collected for 30 min with a micropipette. The Pb concentrations in the xylem sap and in the external solution were determined immediately as described below.
Effect of transpiration on Pb accumulation
The effect of transpiration on Pb accumulation was investigated by adding abscisic acid (ABA) to the solution. The seedlings (15 d old) were exposed to 1 mM Pb(NO3)2 or EDTA–Pb complex (1:1) at pH 5·0 in the absence or presence of 0·1 mM ABA in a 50-mL bottle. The weight of the bottles was recorded before and after the treatment. After 12 h, the plants were sampled as described above and the Pb concentration in the shoots was determined as described below.
Bioassay of Pb toxicity
Six-day-old seedlings were exposed to a 0·5 mM CaCl2 solution containing ionic Pb or EDTA–Pb complexes at molar ratios (Pb:EDTA) of 1:0·5, 1:1, 1:2 and 1:4 for 24 h. These complexes were made by mixing 0·5, 1·0, 2·0 and 4·0 mmol EDTA–Na2–2H2O and 1 mmol Pb(NO3)2 in 1 L of 0·5 mM CaCl2 solution, respectively. The pH was adjusted to 5·0 by 1 N NaOH or HCl. Root length was measured before and after treatments. After the treatment, the roots were washed with 0·5 mM CaCl2 solution three times and the root segments (0–1 cm, 1–2 cm and 2–3 cm back from the root tip) were then excised by a razor blade in a 0·5 mM Ca solution in a Petri dish. Ten root segments were combined and placed into the tubes containing 2 N HNO3. The tubes were left to stand for at least 1 d with occasional shaking. The Pb concentration in the solution was then determined by graphite furnace atomic absorption spectrophotometer (Hitachi Z-5000, Tokyo, Japan).
Staining of intact roots with Fluostain I
Seedlings of sorghum (6 d old) were transferred to a 0·5 mM CaCl2 solution (pH 5·0) containing 0, 1 mM Pb(NO3)2 or 1 mM EDTA–Pb 1:1 complex in the presence of 0·01 % Fluostain I in a 50-mL beaker. Root length was recorded to check the effect of Fluostain I on the root growth at this concentration. At 6 h and 24 h after exposure, the roots were rinsed with deionized water several times. Root was hand-sectioned 1 mm back from the tip with a blade, and then subjected to microscopic observation using an ultraviolet filter set (excitation filter BP 365, dichroitic mirror FT 395, barrier filter LP 397) (Zeiss Axioplan2, Oberkochen, Germany).
Effect of pretreatment with ionic Pb on Pb accumulation
Seedlings (21 d old) were exposed to a 0·5 mM CaCl2 solution (pH 5·0) containing 1 mM Pb(NO3)2 or EDTA–Pb 1:1 complex for 1 d. Half of the seedlings were then transferred to a fresh solution containing 1 mM EDTA–Pb (1:1) for a 1 d. The plants were harvested at day 1 and 2, respectively, as described above. The Pb accumulation during the second day was calculated by subtracting Pb accumulation of plants treated for 1 day from those exposed for 2 d.
Pb uptake from soil amended with EDTA and HNO3
Soil was taken from the experimental farm of Kagawa University as described previously (Ueno et al., 2004). The pH of the soil was 6·0 and the concentration of Pb in the soil solution was 0·01 mg L–1. The soil was sieved (2 mm mesh) and air-dried. Lead as 2PbCO3·Pb(OH)2 was added at a rate of 2500 mg Pb kg–1 soil. Six-day-old seedlings were prepared as described above and were planted in a 1·5-L plastic pot (two seedlings per pot) filled with Pb-enhanced soil. The plants were grown in a greenhouse for 6 weeks. Soil moisture was kept at field capacity with daily addition of tap water. A solution of Na2-EDTA-2H2O or HNO3 was added at a rate of 0·3 mmol kg–1 soil at day 8 before harvest.
Soil solution was collected nondestructively with a looped hollow fibre placed in the pots (Ueno et al., 2004). Briefly, the soil solution sampler system, which consisted of a looped hollow fibre, a silicon tube and a disposable syringe, was set up in a pot. The soil solution was collected in the morning for 2 h once a week before application of chemicals and at 1, 2, 3, 5 and 7 d after addition of chemicals. Approximately 5 mL of solution were collected each time. After collection, the pH and concentrations of Pb were measured immediately as described below.
Determination of Pb concentration
The plant samples were digested in glass tubes containing 5 mL of concentrated HNO3 in a heater (TAITECH, Tokyo, Japan). The temperature started from 70 °C and then increased to 130 °C with intermittent shaking until the samples were completely oxidized. When the digested solution became clear, the sample volume in each tube was reduced to about 1 mL by evaporating the extra HNO3 at 145 °C, and then the tubes were filled with about 20 mL of distilled water. The concentration of Pb in the solution was determined by an atomic absorption spectrometer (Hitachi Z-5000, Tokyo, Japan).
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RESULTS |
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The concentration of Pb in the sorghum shoots was compared between plants exposed to different forms of Pb. The concentration of 1 mM used was commonly found in the soil solution of experiments with EDTA-assisted phytoextraction (e.g. Huang et al., 1997). At this concentration, plant growth did not differ significantly between different treatments due to short exposure. The plants exposed to ionic Pb and EDTA–Pb at 1:0·5 (Pb:EDTA) ratio for 2 d accumulated five times more Pb than those exposed to EDTA–Pb at 1:1 and 1:2 ratios (Fig. 1). When there was excess EDTA (Pb:EDTA, 1:4), the concentration of shoot Pb doubled in comparison with the EDTA–Pb at 1:1 ratio (Fig. 1). The accumulation of Pb in the roots (data not shown) was much higher than that in the shoot, but showed a similar trend to that observed for the shoots.
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The time-course experiment showed that the shoot Pb concentration was very low during the first 6 h in the plants exposed to either ionic Pb or the EDTA–Pb complex at a ratio of 1:1 (Fig. 2A). However, the shoot Pb concentration increased thereafter in the plants exposed to ionic Pb, but not in the plants exposed to EDTA–Pb complex (Fig. 2A). The concentration of Pb in the roots was much higher than that in the shoot (Fig. 2B). Furthermore, the root Pb concentration increased rapidly with exposure time and saturated at 9 h. The Pb concentration in the external treatment solution was also monitored. Twenty-four hours after plants were treated with the EDTA–Pb complex, the concentration of total soluble Pb in the external solution increased compared with that of the initial solution (data not shown).
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The concentration of Pb in the xylem sap was measured in the plants exposed to 1 mM ionic Pb or EDTA–Pb (1:1). Xylem sap was not available for the plants exposed to ionic Pb because of plant toxicity (see below). The Pb concentration in the xylem sap increased with the exposure time to EDTA–Pb, but the concentration was only 0·04 µM at 8 h, which was about 1/25 000 of the concentration in the external solution (Fig. 3).
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The effect of transpiration on Pb accumulation was examined by adding ABA to the treatment solution. ABA is known to suppress transpiration. In the presence of 0·1 mM ABA, transpiration during 12 h decreased by 41·5 % and 37·5 %, respectively, in the plants exposed to ionic Pb and EDTA–Pb (1:1) (Fig. 4A). The Pb concentration in the shoots also decreased corresponding with the decreased transpiration in plants exposed to either Pb form (Fig. 4B).
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The toxicity of ionic or EDTA-complexed Pb was assessed using root elongation. Root elongation was inhibited with 1 mM ionic Pb by 90 % (Fig. 5). In contrast, Pb complexed with EDTA at 1:1 and 1:2 molar ratios did not inhibit root elongation (Fig. 5). At 1:4 ratio of Pb to EDTA, root elongation was also severely inhibited. Root elongation of sorghum exposed to EDTA–Pb at the mole ratio of 1:0·5 (Pb:EDTA) was also inhibited, suggesting that the concentration of unchelated Pb in the solution was high enough to be toxic. The effect of EDTA alone on root elongation in sorghum was also investigated. Root elongation was inhibited by 38 % and 82 % by 2 mM and 4 mM EDTA, respectively, suggesting that EDTA was also toxic to plant roots at high concentrations (data not shown).
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The Pb content in the different root segments was determined. Over all, the Pb content in the roots exposed to ionic Pb was much higher than that in the roots exposed to EDTA–Pb complex at a molar ratio of Pb:EDTA higher than 1 (Table 1). For example, the root apices (0–1 cm) exposed to ionic Pb contained 41·7 nmol root segment–1, while those exposed to EDTA–Pb (1:1) contained only 22·9 pmol root segment–1, which was almost 1/2000 of that exposed to ionic Pb (Table 1). In the roots exposed to ionic Pb, more Pb accumulated in the root tips of 0–1 cm than in the mature regions (>2 cm), suggesting that the root apex is the first target of Pb toxicity.
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The roots were stained with the Fluostain I dye, which shows a pale blue fluorescence after binding to the cellulose of the cell walls (Hughes and McCully, 1975). Therefore, this dye can be used as a tracer of apoplastic solute flow in the roots. The presence of this dye at 0·01 % and 0·001 % has no effect on root elongation (data not shown). Fluorescence was only observed at the surface of the epidermal cells in the roots in the control treatment (no Pb or EDTA–Pb) or in those exposed to EDTA–Pb (1:1) (Fig. 6), suggesting that the dye was stopped at the root surface and cannot enter the stele. In contrast, in the roots exposed to ionic Pb, fluorescence was observed not only in the epidermal cells but also in the whole cortex and central stele as early as 6 h after exposure (Fig. 6). At 24 h, the roots exposed to ionic Pb became too soft for sectioning due to loss of vigour (Fig. 6).
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When plants exposed to either ionic Pb or EDTA–Pb (1:1) for 1 d were transferred to a solution containing EDTA–Pb (1:1), an enhanced accumulation of Pb the next day was observed in the plants pre-treated with ionic Pb only (Fig. 7). This result suggests that ionic Pb damaged the physiological barrier in the root and facilitated the transport of EDTA–Pb into the stele.
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Pb accumulation from different forms was further examined in a Pb-contaminated soil. Application of EDTA or HNO3 to the soil 8 d before harvest resulted in different Pb concentrations in the soil solution and in shoot accumulation. The concentration of Pb in the soil solution was much higher after addition of EDTA than HNO3 (Fig. 8A). However, the accumulation of Pb was much lower in plants grown on soil with added EDTA than those with added HNO3 (Fig. 8B). These results indicate that EDTA was very effective in solubilizing Pb in soil, but only a small fraction of the solubilized Pb was taken up.
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DISCUSSION |
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Because of a physiological barrier in the roots, metals cannot reach the stele freely. For a metal to be accumulated in the shoots from the soil environment, at least three steps are required. The first one is the transport of a metal from soil to the surface of root cells via diffusion or mass flow. The second step is the transport of a metal from the outside (rhizosphere) to the root cells and the third one is the release of a metal into the xylem (xylem loading) (Mitani and Ma, 2005). Transport proteins are involved in the latter two processes. The present study investigated whether a transport system exists for EDTA–Pb in sorghum. The present results show that plant roots do not possess a transporter for the EDTA–Pb complex based on the following evidence. First, the short-term uptake experiment showed that the uptake of Pb was greatly reduced when EDTA was present (Figs 1 and 2). Two kinds of transporters for cations, ionic and chelated forms, have been reported depending on metals and plant species. Most cations such as Zn, Fe and Cu are transported in the ionic forms (Kochian, 2000), and it is generally believed that the chelated form of metals are less available for uptake as compared with the ionic ones. However, in gramineous plants, Fe is transported in a chelated form (Fe–phytosiderophore complex) (Murata et al., 2006). Low uptake from EDTA–Pb suggests that a transporter is not involved. Secondly, the Pb concentration in the xylem sap of plants exposed to EDTA–Pb was only 1/25 000 of the external solution (Fig. 3). This result is in agreement with the recent finding by Schaider et al. (2006). They found that under normal conditions, xylem sap solute concentrations from EDTA–metal complexes are relatively low (i.e. <0·5 % of concentration in solution) in Brassica juncea. If a transporter is involved, the concentration in the xylem sap is usually higher than that in the external solution. For example, Al in the xylem sap reached 4-fold higher concentration than that in the external solution as early as 2 h after exposure in buckwheat (Ma and Hiradate, 2000). Thirdly, the concentration of total soluble Pb in the external solution of the EDTA–Pb treatment increased with time, suggesting that uptake of EDTA–Pb, if any, was slower than uptake of water. This means that there is a lack of active uptake of EDTA–Pb by the roots. Usually, a depletion of a metal in the external solution is observed if its uptake is mediated by a transporter. Fourthly, the Pb uptake from EDTA–Pb depended on transpiration (Fig. 4), which suggests that the EDTA–Pb complex is transported passively and that transpiration is the driving force for Pb translocation.
The higher accumulation from ionic Pb can be attributed to root damage. Ionic Pb inhibited root elongation (Fig. 5). Root staining showed that there is a physiological barrier on the epidermal cells of the root tips, which may prevent solutes from passing freely through the cortex into the stele (Fig. 6, controlled roots), although the barrier has not been identified. Exposure to ionic Pb caused damage of the physiological barrier (Fig. 6), resulting in a free flow of Pb to the shoot. This is supported by the findings that Pb accumulation is controlled by transpiration (Fig. 4) and that Pb accumulation in the shoot dramatically increased after 6 h in the plants exposed to ionic Pb (Fig. 2A). It is also supported by the finding that pre-treatment with ionic Pb enhances the subsequent accumulation from EDTA–Pb (Fig. 7). In rice, it was also reported that the root structure was damaged by excessive Na, resulting in enhanced flow of Na into the xylem vessels and increased accumulation of Na in the shoot (Ochiai and Matoh, 2002).
In contrast to ionic Pb, EDTA–Pb was not toxic to sorghum roots at the level investigated (Fig. 5) and the physiological barrier was also not damaged in the roots (Fig. 6). This result is consistent with a previous study with Indian mustard (Vassil et al., 1998). In their study, phytotoxicity of Pb was observed when the concentration of EDTA was lower than that of Pb in the treatment solution. The toxicity of Pb is suggested to be caused by the binding of Pb to cellular components (Geebelen et al., 2002). Chelation of Pb by EDTA prevents this binding, therefore detoxifying Pb. This is supported by the finding that a lower concentration of Pb was detected in the roots exposed to EDTA–Pb than those exposed to ionic Pb (Table 1).
Previous studies reported that the accumulation of Pb was greatly enhanced by application of EDTA in soil (e.g. Blaylock et al., 1997; Huang et al., 1997). In an ultrastructural study of chelated Pb transport in Chamaecytisus proliferus spp. Proliferus ‘palmensis’ grown in a solution, the Pb transported from roots to shoots increased in the EDTA–Pb treatment (Jarvis and Leung, 2001). Indian mustard plants exposed to Pb and EDTA in nutrient solution were able to accumulate up to 55 mmol kg–1 Pb in dry shoot tissue [1·1 % (w/w)] (Vassil et al., 1998). They suggested that the soluble EDTA–Pb complex is transported through the plant, via the xylem, and accumulates in the leaves. However, this high Pb accumulation may be attributed to the excess use of EDTA. Excessive EDTA causes toxicity to the root and shoot growth (Fig. 5 and Vassil et al., 1998). It may also disrupt the physiological barriers including the Casparian strip (Nowack et al., 2006). In fact, there is a threshold of EDTA concentration to ‘induce’ the accumulation of high Pb (Vassil et al., 1998). At a ratio of EDTA–Pb lower than 1, no EDTA was detected in the shoot. When the ratio of EDTA to Pb was higher than 2, an increase in EDTA was observed in Indian mustard with a concomitant decrease in shoot growth. These aforementioned observations support the interpretation that the apparent accumulation of Pb, observed by Vassil et al. (1998), was caused by the toxicity of excessive EDTA. Excessive EDTA may remove Zn and Ca ions in the plasma membranes, which are essential for stabilizing the membranes, thereby destroying the physiological barriers of the roots. EDTA can also chelate various essential divalent cations such as Fe, Zn and Cu. Therefore, excessive presence of EDTA may disrupt the biochemistry of the leaf cells and ultimately cause cell death (Vassil et al., 1998).
Two major limiting factors in Pb phytoextraction from contaminated soils are the low bioavailability of Pb in the soil and the low Pb translocation from roots to shoots. Although EDTA is an efficient chelator of Pb, the present results show that plant roots do not possess a system for transporting EDTA–Pb. Although excessive use of EDTA can enhance the accumulation of Pb to some extent, there is an environmental risk of EDTA due to its low biodegradability. Therefore, for an efficient phytoextraction of Pb, approaches which facilitate the uptake of the EDTA–Pb complex are required. From the results of the soil experiment reported here, it seems that lowering soil pH is more effective for Pb accumulation in the plant than applying EDTA to the soil (Fig. 8).
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ACKNOWLEDGEMENTS |
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This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 13556010 to J. F. Ma and by CAS International Partnership Project (CXTD-Z2005-4)). We thank Lawrence Datnoff and Fangjie Zhao for their critical reading of this paper.
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