Gibberellic Acid, Kinetin, and the Mixture Indole–3-Acetic Acid–Kinetin

Environ. Sci. Technol. 2007, 41, 8165–8170

Gibberellic Acid, Kinetin, and the Mixture Indole–3-Acetic Acid– Kinetin Assisted with EDTA-Induced Lead Hyperaccumulation in Alfalfa Plants MARTHA L. LÓPEZ,† JOSÉ R. PERALTA-VIDEA,‡ JASON G. PARSONS,‡ TENOCH BENITEZ,‡ A N D J O R G E L . G A R D E A - T O R R E S D E Y * ,†,‡ Environmental Science and Engineering Ph.D. Program and Chemistry Department, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968

Received June 12, 2007. Revised manuscript received August 28, 2007. Accepted September 11, 2007.

There are a few plant species considered potential hyperaccumulators for heavy metals, particularly lead (Pb). In this study, alfalfa plants grown in hydroponics were exposed to Pb at 40 mg/L, ethylenediaminetetraacetic acid (EDTA) equimolar to Pb, and 1, 10, and 100 µM concentrations of the phytohormones indole–3-acetic acid (IAA), gibberellic acid (GA), and kinetin (KN) and a mixture of IAA and KN at 100 µM each. Metal quantification by inductively coupled plasma/optical emission spectroscopy demonstrated that plants treated with Pb/EDTA plus KN at 1, 10, and 100 µM increased the Pb concentration in alfalfa leaves (compared to Pb alone) by factors of 17, 43, and 67, respectively, and by factors of 2, 5, and 8, respectively, compared to the Pb/EDTA treatment. The correlation coefficient between the Pb concentration in leaves and the concentrations of KN in the medium was 0.9993. In addition, the leaves of plants exposed to a Pb/EDTA/100 µM IAA–KN mixture had approximately 9500 mg of Pb/kg of dry weight, demonstrating that non-Pb hyperaccumulating plants could hyperaccumulate Pb when treated with EDTA and a mixture of IAA–KN. The X-ray absorption spectroscopic studies demonstrated that the absorption and translocation of Pb was in the same oxidation state as the supplied Pb(II).

Introduction The excess of lead (Pb) in the environment has been a major concern in recent years, mainly in industrialized countries (1). Pb is a toxic heavy metal that causes numerous health problems. The toxicity of Pb has been associated with the damage of some biochemical processes in mammals. In humans, Pb has been linked to developmental and behavioral problems and severe damage to the nervous system (2, 3). The removal of Pb from soil has been carried out through chemical, physical, and thermal processes such as vitrification, stabilization, and chemical oxidation, among others (4, 5). However, some of these technologies entail high costs * Corresponding author phone: (915) 747-5359; fax: (915) 7475748; e-mail: [email protected]. † Environmental Science and Engineering Ph.D. Program. ‡ Chemistry Department. 10.1021/es0714080 CCC: $37.00

Published on Web 10/13/2007

 2007 American Chemical Society

and are invasive to local ecosystems. Phytoremediation is the use of plants for the removal of toxic substances from places containing widespread contamination at low or medium levels (6). Suitable plants for phytoremediation purposes are those called hyperaccumulators. So far, there are several criteria for the classification of hyperaccumulating species: (a) a plant must accumulate 0.1% of the element in its dry leaf tissue (7, 8), (b) the metal content in shoots should be 10–500 times higher than the metal content found in regular plants (9), (c) the metal concentration in shoots needs to be greater than the metal concentration in roots (10, 11), and (d) the enrichment coefficient needs to be greater than 1 (12, 13). The uptake of elements by plants depends on the availability of the element, the pH of the media, the interactions with other elements, and the species of plant, among other factors. Pb is considered to have low availability for plant uptake because it precipitates in the presence of carbonates and phosphates. These are chemicals commonly found in the rhizosphere of plants. Furthermore, Pb is immobilized in soil when it forms complexes with the organic matter (14). In an investigation performed in hydroponics by Wierzbicka (15), it was found that certain plant species are more tolerant to Pb toxicity. This investigator found that Maidenstears (Silene vulgaris), Buckler Mustard (Biscutella laevigata), and Rough hawkbit (Leontodon hispidus) grew well in a medium containing 5.0 mg of Pb/L. Wierzbicka (15) classified these species as Pb tolerant. Researchers have found that certain chelating agents such as ethylenediaminetetraacetic acid (EDTA) can increase the solubility of Pb, increasing sometimes its uptake and translocation from roots to shoots (16–18). Sarret et al. (18) found that EDTA helps to increase the availability of Pb and is found in leaf tissues complexed with Pb. In another investigation, de la Rosa and collaborators (19) found that, in tumbleweed (Salsola tragus) plants grown in hydroponics, Pb was transported and distributed in leaf tissues as a Pb/EDTA complex. However, EDTA by itself does not promote the uptake and translocation of enough Pb for a plant to be considered as a Pb hyperaccumulating species. Several studies have demonstrated that alfalfa (Medicago sativa L.) plants are able to uptake heavy metals from soil and water (20, 21). However, alfalfa is not considered to be a heavy-metal hyperaccumulating species. In a previous investigation, seedlings of alfalfa were grown in a hydroponic solution containing 40 mg of Pb/L and the growth-promoting hormone indole–3-acetic acid (IAA) at 1, 10, and 100 µM (22). The results demonstrated that the combination of 100 µM IAA and 0.2 mM EDTA increased the Pb accumulation in leaves by a factor of 30, compared to the Pb concentration in leaves of plants exposed to Pb alone and by a factor of 7 compared to the Pb concentration in leaves of plants exposed to Pb/EDTA. The objectives of the present study were to determine the effects of the growth hormones IAA, gibberellic acid (GA), and kinetin (KN), alone and combined with the chelating agent EDTA, on plant growth and the uptake and translocation of Pb. These phytohormones regulate plant growth and development (23), and it is hypothesized that they will increase the Pb uptake and translocation. Also, an experiment was set to determine the effectiveness of the mixture IAA–KN (alone and combined with EDTA) on Pb uptake and translocation. These phytohormones were selected based on preliminary results. In this study, the element uptake data were obtained by inductively coupled plasma/ optical emission spectroscopy (ICP/OES). In addition, X-ray absorption spectroscopy (XAS) was used to determine the oxidation state through X-ray absorption near-edge structure VOL. 41, NO. 23, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

8165

(XANES). A linear-combination XANES (LC-XANES) fitting was performed in several samples and model compounds in order to have an idea about the possible coordination environment of the Pb taken up by roots, stems, and leaves of alfalfa exposed to the different treatments.

Materials and Methods Seeds of alfalfa (M. sativa L., Mesa variety) were obtained from California Crop Improvement Association. The seeds were immersed for 5 min in 100% ethanol and rinsed with sterilized deionized water (DI). Subsequently, the seeds were immersed in a 4% sodium hypochlorite solution for 30 min with stirring. The seeds were then rinsed with sterilized DI and placed in paper towels soaked with an antibiotic–antimycotic solution (Sigma A5955, St. Louis, MO) to avoid fungal and bacterial contamination. For germination, the seeds were incubated in the dark for 4 days and then exposed to light for 1 day. After that, 50 seedlings were transferred to wide-mouth 200-mL Mason jars containing 150 mL of a lowphosphate Hoagland nutrient solution (24). Sets of 5 seedlings were packed in plastic tubes, and the tubes were inserted into a holed polystyrene lid, allowing the full contact of the roots with the nutrient solution. Two experiments were performed. In the first experiment, treatments contained 40 mg of Pb/L, IAA, GA, or KN at 0, 1, 10, and 100 µM and EDTA equimolar to Pb (0.2 mM). The treatments were (1) control (C), (2) 1 µM hormone (H), (3)10 µM H, (4) 100 µM H, (5) Pb, (6) Pb/1 µM H, (7) Pb/10 µM H, (8) Pb/100 µM H, (9) Pb/ EDTA, (10) Pb/EDTA/1 µM H, (11) Pb/EDTA/10 µM H, and (12) Pb/EDTA/100 µM H. In the second experiment, treatments contained 40 mg of Pb/L, the mixture of IAA–KN at 100 µM each, and EDTA at 0.2 mM. Treatments were (1) C, (2) IAA–KN, (3) Pb, (4) Pb/IAA–KN, (5) Pb/EDTA, and (6) Pb/EDTA/IAA–KN. Controls in each experiment were plants grown in the low-phosphate Hoagland nutrient solution. In all cases, the solution pH was adjusted to 5.4 using NaOH or HNO3 as needed. In both experiments, each treatment was replicated four times for statistical purposes. The jars were allowed to set for 2 weeks at 25 ( 2 °C, a light/dark cycle of 12/12 h, and an irradiation of 53 µmol/(m2 s). The system was continuously aerated using aquarium pumps. After the growth period, 10 plants were randomly selected from each replicate/treatment and measured from the main root apex to the crown and from the crown to the main shoot apex to determine the effects of the treatments on plant growth. For the Pb determination, a sample of 20 plants from each replicate/treatment was washed with 0.01 M HNO3, rinsed with DI, sectioned in roots, stems, and leaves, and dried in a Fisher Scientific Isotemp oven at 60 °C for 3 days. Samples were then digested in a CEM Marsx microwave oven (CEM Corp., Mathews, NC) with a 5-mL trace of pure HNO3 (SCP Science, Champlain, NY) and diluted to 15 mL using double DI. The Pb concentration in roots, stems, and leaves was determined using an inductively coupled plasma optical emission spectrometer Optima 4300 DV (Perkin-Elmer, Shelton, CT). For quality check/quality analysis of the ICP readings, for every 10 samples the blank and a spiked sample containing Pb at 0.05 mg/L were read. The accuracy of the instrument was (0.010%. XAS Sample Preparation. The XAS spectra were obtained from roots, stems, and leaves of the Pb-treated plants. The samples were frozen in liquid nitrogen for 45 min and lyophilized to remove any free water using a Freezone 4.5 freeze dryer at -45 °C and 70 × 10-3 Mbar (Labconco, Kansas City, MO). The samples were ground, homogenized, and loaded into 1.0-mm aluminum sample holders with Kapton tape windows. The XAS studies were performed at Stanford Synchrotron Radiation Laboratory (SSRL; Palo Alto, CA). XAS Data Collection. The X-ray absorption spectra were collected on the Pb LIII edge (13.035 keV) on beamline 2–3 8166

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 23, 2007

with a beam current ranging between 80 and 100 mA, an energy of 3.0 GeV, and a Si [220, φ (angle) 90° orientation] double-crystal monochromator with a 1.0-mm slit. The monochromator crystal was detuned by 30% to reject higher order harmonic oscillations. The spectrum of an internal Pb(0) foil was also collected with the samples for calibration purposes. A Canberra 13-element germanium detector (Canberra Instruments, Meriden, CT) was used to collect the fluorescence spectra of the Pb-laden plant samples. The model compounds were run in transmission mode. The model compounds lead(II) acetate, lead(II) citrate, and lead(II) nitrate were diluted to 5% by mass in boron nitride using mortar and pestle to obtain a homogeneous mixture. The dilution of the model compounds was performed to obtain a one-absorption-unit change across the absorption edge. All samples were run at ambient temperature. XAS Data Analysis. The samples were analyzed using standard data reduction processes and the WinXAS software (25, 26). The data were first calibrated based on the first inflection point of the Pb(0) foil (E0 13.035 keV) using a second-degree derivative of the absorption edge. After energy calibration, the samples were background-corrected using a 1° polynomial fitting to the pre-edge region and normalized to one absorption unit. After normalization, the XANES spectra of samples and model compounds were then extracted by sectioning the XAS spectra from 12.80 to 13.20 keV for comparison purposes. Statistical Analysis. The growth and uptake data were analyzed with one-way analysis of variance (ANOVA) using the SPSS software version 11.0 (SPSS Inc., Chicago, IL). The significant differences between treatment means were detected using the Tukey-HSD (honestly significant difference) test. Any reference to a significant difference between data is based on a probability of P < 0.05, unless otherwise stated.

Results and Discussion Effect of IAA, GA, KN, EDTA, and Pb(II) on Alfalfa Root Growth. The data showed that the roots of plants exposed to IAA at 1 µM (6.5 cm) and 10 µM (5.0 cm) were significantly longer compared to control roots (plants exposed only to modified Hoagland’s medium measured as 4.0 cm) (Figure 1a in the Supporting Information). However, plants exposed to IAA at 100 µM, Pb at 40 ppm, Pb/IAA at 1, 10, and 100 µM, and Pb/EDTA/IAA at 100 µM produced significantly shorter roots (between 2.8 and 2.2 cm). Reports indicated that the effects of IAA on root growth are inversely related to its concentration in the growth media (27). It has been documented that IAA at high concentrations can increase the biosynthesis of ethylene, which reduces root elongation (28). Spiro et al. (29) reported that, in cucumber (Cucumis sativus), IAA at a concentration as low as 1 µM reduced the elongation of the roots. In the present study, Pb/EDTA and Pb/EDTA/ IAA at 10 µM also significantly increased the growth of roots (5.5 and 5.0 cm, respectively). These data suggest that the increase in cell permeability produced by EDTA plus the increase in nutrient translocation promoted by IAA overcame the toxic effect produced by Pb (30). There are many reports on the positive effect of EDTA for the accumulation of nutrients (31). However, to our knowledge, there are no reports on the effects of EDTA and phytohormones on plant growth. On the other hand, GA and KN did not have a significant effect on the elongation of alfalfa roots. The size of the roots was maintained between 3 and 4 cm in all treatments. Tanimoto (27) reported that GA applied at different concentrations on many plants did not produce any significant change in the root length. Effect of IAA, GA, KN, EDTA, and Pb(II) on Alfalfa Shoot Growth. None of the treatments produced significant effects on the elongation of alfalfa shoots (Figure 1b in the Supporting Information). The stem length was recorded in the range of

FIGURE 1. Pb concentration in (a) roots, (b) stems, and (c) leaves of alfalfa plants treated with Pb at 40 mg/L, EDTA equimolar to Pb, GA, and KN. The numbers stand for (1) Pb alone (box with vertical lines), (2) Pb/1 µM H, (3) Pb/10 µM H, (4) Pb/100 µM H, (5) Pb/EDTA (solid box), (6) Pb/EDTA/1 µM H, (7) Pb/EDTA/10 µM H, and (8) Pb/EDTA/100 µM H. Error bars represent SE. 3.5–5.0 cm in all treatments. It seems that, even at high concentrations, GA, IAA, and KN do not inhibit shoot elongation (27). Effect of a Mixture IAA–KN, EDTA, and Pb(II) on Alfalfa Root and Shoot Growth. It has been reported that there is a reduction upon KN degradation in the IAA overproducing tobacco plant (28). However, the results found in the present investigation have shown that exogenous KN at the same concentration of exogenous IAA overcame the effects produced by IAA on plant growth. In this study, only the treatment of Pb/EDTA (treatment 5) produced significantly larger shoots. Reports indicated that corn (Zea mays) plants grown in soil and treated with EDTA, had more phosphorus than control plants (32). Alfalfa is a plant that requires high phosphorus intake (33), which could be responsible for the larger shoots. Pb(II) Uptake by Alfalfa Plants Exposed to KN and GA. Previous results demonstrated that IAA combined with EDTA increased the Pb translocation from the roots to the leaves by a factor of 30 compared to plants exposed to Pb alone (22). The effects of GA and KN, alone and combined with EDTA, on the Pb uptake and translocation are shown in Figure 1. Figure 1a shows that the roots of plants deprived of EDTA (treatments 1–4) had significantly more Pb than the plants treated with EDTA (treatments 5–8). In addition, the Pb concentration in roots of plants exposed to treatment 4 (Pb/ 100 µM KN) had significantly more Pb (around 104000 mg of Pb/kg of dry weight biomass, DWB) compared to treatments 1–3 (GA at 1, 10, and 100 µM and KN at 1 and 10 µM). It is known that KN at high concentrations (100 µM) induces

cytokinesis (cell division) and the growth of plants (34). This suggests that the increase in the number of cells increased the Pb uptake capacity of the roots. Figure 1a also shows that the Pb concentration in roots from all treatments containing EDTA was negligible. The decrease in metal concentrations in roots of plants treated with EDTA might be due to the fact that metal chelate complexes avoid metal sorption to the cation-exchange sites in the apoplast (35). The data shown in parts b and c of Figure 1 suggest that Pb was mobilized from roots to stems and leaves. As shown in Figure 1b, plants exposed to either GA or KN at 10 µM had less concentration of Pb when the medium was deprived of EDTA. The highest Pb concentrations were found in stems of plants treated with 1 µM GA and 1 µM KN, approximately 15% (8230 mg/kg of DWB) and 18% (7980 mg/kg of DWB) lower, respectively, compared to the Pb concentration in stems of plants treated with Pb alone (9702 mg/kg of DWB). Contrary to the results observed in roots and stems, the concentration of Pb in alfalfa leaves was significantly higher in all treatments containing EDTA. The leaves of plants treated with Pb only had 55 mg of Pb/kg of DWB. However, the leaves of plants treated with Pb/EDTA (treatment 5) had 309 mg of Pb/kg of DWB, which represents an increment by a factor of 6. It has been documented elsewhere that ions can be transported across cell walls and intercellular spaces by passive transport in the apoplast. Tanton and Crowdy have shown that the Pb/EDTA complex is transported through the transpiration stream, which transports nutrients from roots to leaves through the apoplast in roots where the Casparian strip is not fully developed (36). The Pb concentration in leaves when the medium contained GA at 1 µM increased to 462 mg/kg of DWB, about 8 times higher compared to the Pb concentration in leaves of plants treated with Pb alone. More research is needed to explain the reduction in the Pb concentration in leaves treated with GA at 10 and 100 µM. As shown in Figure 1c, the highest Pb translocation from roots to leaves was found in plants exposed to EDTA plus KN at 1, 10, and 100 µM. The figure shows that the Pb concentration in leaves increased as the concentration of KN in the medium increased. Simple calculations have shown that the increases (with respect to the Pb alone treatment) were by factors of 17, 43, and 67, respectively. The increases in Pb concentrations with respect to Pb/EDTA were by factors of 2, 5, and 8, respectively. A correlation analysis for the Pb concentrations in leaves and the concentration of KN in the medium has shown an r value of 0.9993. The data showed that KN did not promote root and shoot enlargement, which indicates that other physiological pathways were activated. It has been reported that cytokinins are able to promote photosynthesis and to retain photosynthate (37), which could explain the plants’ ability to translocate more Pb to the leaves. Sayed et al. (37) reported that, in Safflower (Carthamus tinctorius L.) plants exposed to Pb(NO3)2 and KN, there was a reduction in Pb toxicity due to an increase in the leaf membrane stability and chlorophyll content. Uptake of Pb by Alfalfa Plants Exposed to a Mixture of IAA–KN and EDTA. Reports have indicated that the combination of IAA and KN can increase cell division and the growth of cultured cells (30). On that basis and on the preliminary results obtained with KN and IAA on Pb uptake and translocation, an experiment was set using these phytohormones at 100 µM each. Figure 2a shows that the concentration of Pb in roots was significantly higher in plants treated with hormones without EDTA. However, the concentration of Pb in roots (about 95000 mg of Pb/kg of DWB) was lower compared to the one found with KN only (104000 mg of Pb/kg of DWB; Figure 1a). As in the previous experiment, EDTA did not increase the concentration of Pb in roots and the amounts were significantly lower compared VOL. 41, NO. 23, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

8167

FIGURE 3. Pb LIII edge XANES (12.9–13.2 keV) for (a) Pb(II), (b) Pb(II)/EDTA, and (c) Pb(II)/EDTA/hormones (IAA, GA, and KN). The spectra correspond to roots, stems, and leaves of alfalfa and the model compounds Pb foil [Pb(0)], lead citrate, Pb/EDTA solid, and Pb/IAA/EDTA solution. FIGURE 2. Pb concentration in (a) roots, (b) stems, and (c) leaves of alfalfa plants treated with Pb at 40 mg/L, EDTA equimolar to Pb, and the mixture IAA–KN at 100 µM each. The numbers stand for (1) Pb, (2) Pb/IAA–KN, (3) Pb/EDTA, and (4) Pb/EDTA/IAA–KN. Error bars represent SE. to those in the treatments without EDTA. More studies are required to explain these results. Figure 2b shows that the Pb accumulation in stems (about 25000 mg/kg of DWB) was numerically higher in plants exposed to Pb alone. The concentration of Pb accumulated using EDTA was significantly lower and very similar with/ without hormones. Figure 2c shows that the concentration of Pb in plants exposed to Pb/EDTA/IAA–KN (about 9500 mg of Pb/kg of DWB) was higher by a factor of 110 compared to the Pb concentration found in leaves of plants treated with Pb/EDTA without phytohormones. Comparing Figures 1c and 2c, one can see that the concentration of Pb in leaves of plants treated with Pb/EDTA/IAA–KN at 100 µM each was 2-fold compared to the Pb accumulation from Pb/EDTA/KN at 100 µM and about 3-fold compared to the accumulation from the treatment Pb/EDTA/IAA at 100 µM (22). These results have shown for the first time that the combination of IAA and KN assisted by EDTA significantly increased the translocation of Pb from roots to leaves. The concentration of Pb in shoots of alfalfa treated with Pb/EDTA/IAA–KN satisfied at least three criteria to consider that this treatment made alfalfa a Pb-hyperaccumulating plant. According to Altamura et al. (38), KN combined with IAA increases the protein of the gene RolB in tobacco plants. The protein of RolB gene is able to induce root and shoot formation. In addition, IAA and KN also showed to increase the weight and number of microtubers inside potato (Solanum tuberosum L.) plants, also increasing the ion transportation inside plants. IAA has been related to the increase in the tuber size, whereas KN has been related to the increase in the number of tubers 8168

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 23, 2007

(39). An increase in the protein content, the number and size of microtubers, and the detoxifying effect of EDTA is maybe responsible for the increase in Pb translocation from the roots to the leaves. XAS Results. Spectra of the model compounds {Pb foil [Pb(0)], Pb/EDTA solid, Pb/EDTA solution, and lead citrate [Pb(II)]} and Pb-loaded roots, stems, and leaves of alfalfa plants are shown in Figure 3a–c. The spectra of lead citrate and Pb foil are used to show the edge energy and the oxidation state of different Pb compounds. In addition, the LC-XANES fittings were performed with the model compounds supplied in Table 1. Figure 3a shows the spectra of Pb in roots, stems, and leaves and the spectra of the Pb foil and lead citrate. The spectra of Pb in plant samples are very similar to the spectrum of the model compound lead citrate, which indicates that Pb in plant tissues was in the same oxidation state as the supplied Pb(II). In addition, these data also indicated that Pb was complexed into a citrate-type complex or coordinated to small organic acids, as has been shown in the literature (40, 41). Figure 3b shows Pb/EDTA-type spectra in all tissues of plants grown in media containing Pb/EDTA. Sarret et al. (18) reported that, in common bean (Phaseous vulgaris) exposed to EDTA/Pb in hydroponics, a percentage of the absorbed Pb was found as the Pb/EDTA complex within the leaf tissue. However, in the absence of EDTA, Pb has been found to be complexed into a lead carbonate system (18). Figure 3c shows that Pb/EDTA solid and Pb/EDTA/IAA solution have similar spectra, which suggested that the addition of phytohormones did not change the oxidation state or the coordination environment of Pb in the Pb/EDTA/hormone complex. As observed in Figure 3c, regardless of the presence of phytohormones, all tissues show Pb spectra similar to the spectrum of the Pb/EDTA complex, suggesting that plants might absorb and transport Pb as the complex Pb/EDTA.

TABLE 1. LC-XANES Fittings from Roots, Stems, and Leaves of Alfalfa Plants Treated with Pb, Pb/EDTA, and Pb/EDTA/IAA Using the Model Compounds Pb/EDTA, Lead(IV) Acetate, Lead(II) Acetate, Lead Nitrate, and PbS sample alfalfa Pb roots alfalfa Pb stems alfalfa Pb leaves alfalfa Pb/EDTA roots alfalfa Pb/EDTA stems alfalfa Pb/EDTA leaves alfalfa Pb/IAA roots alfalfa Pb/IAA stems alfalfa Pb/IAA leaves alfalfa Pb/IAA/EDTA roots alfalfa Pb/IAA/EDTA stems alfalfa Pb/IAA/EDTA leaves

% Pb/ % Pb % Pb % Pb EDTA (C3H5O2)4 (C3H5O2)2 (NO3)2 20.4 10.5 3.5 17.6 8.3 0.0 13.5 14.5 0.0

% PbS

66.2 64.3 62.7 78.3 91.7 94.0 73.7 66.0 68.1

8.7 2.9 9.7 0.0 0.0 6.0 0.0 4.2 0.0

4.7 0.0 0.0 0.0 0.0 0.0 10.8 0.0 2.7

0.0 22.4 24.2 4.0 0.0 0.0 1.95 15.3 29.2

61.4

3.3

0.0

18.9 16.3

76.7

5.3

0.0

0.0 17.9

63.1

2.9

2.6

3.5 27.9

Table 1 shows the LC-XANES fittings from roots, stems, and leaves of alfalfa plants treated with Pb, Pb/EDTA, and Pb/EDTA/IAA using model compounds Pb/EDTA, lead(IV) acetate, lead(II) acetate, lead nitrate, and PbS. Table 1 corroborated that, in Pb, Pb/IAA, and Pb/EDTA/IAA treatments, Pb was mainly bound to the plant tissues in a structure similar to that of the model compound Pb/EDTA (60–70%). Table 1 also shows that in tissues of plants treated with Pb/ EDTA almost 80–90% of the Pb was present as Pb/EDTA. The LC-XANES fittings (Table 1) suggest that alfalfa plants transformed the initial Pb(NO)3 supplied to the medium (without EDTA) into a Pb complex similar in geometry to the Pb/EDTA complex. The residual Pb remained mainly bound in structures similar to lead nitrate in roots of all treatments. In addition, stems and leaves of alfalfa plants showed a high percent of Pb bound in the same structure as that of lead sulfide with the exception of the Pb/EDTA treatment. Sharma et al. (42) found that LC-XANES fittings from rattlebush plants (Sesbania drummondii) exposed to 500 mg/L of Pb revealed that Pb was present in structures similar to those of lead acetate, lead nitrate, and lead sulfide, suggesting that Pb is transported in several complexes inside the plants. Other studies have shown that Pb becomes complexed to organic acids for storage within the plants to minimize health effects (25, 26). The relative stabilities of the Pb complexes have been reported through the galvanostatic method, which showed that the Pb/EDTA complex is much stronger than lead citrate type complexes (43). Further studies are needed to determine the biochemical mechanism(s) used by the plants to absorb and mobilize the complex Pb/EDTA.

Acknowledgments The authors acknowledge the HBCU/MI Environmental Technology Consortium that is funded by the Department ofEnergy,TheNationalInstitutesofHealth(GrantS06GM801233), and the EPA/SCERP program. J.L.G.-T. acknowledges the STAR program of the UT System and the Dudley family for the Endowed Research Professorship. The authors also acknowledge the Stanford Synchrotron Radiation Laboratory through the DOE-funded Gateway Program. M.L.L. also acknowledges the Consejo Nacional de Ciencia y Tecnologia of Mexico (CONACyT) for its financial support (Grant 178763).

Supporting Information Available Figure showing root and stem lengths of alfalfa plants grown with Pb, IAA, GA, KN, and EDTA. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Xiong, Z. T. Lead uptake and Effects on Seed Germination and Plant Growth in a Pb Hyperaccumulator Brassica pekinensis Rupr. Bull. Environ. Contam. Toxicol. 1998, 60, 285–291. (2) DeMichelle, S. J. Nutrition of Lead. Comp. Biochem. Physiol. 1984, 78 (A), 401–408. (3) Aung, N. N.; Yoshinaga, J.; Takahashi, J. Exposure Assessment of Lead among Japanese Children. Environ. Health Prev. Med. 2004, 9, 257–261. (4) Cunningham, S. D.; Shan, J. R.; Crowley, D. E.; Anderson, T. A. Phytoremediation of contaminated water and soil. ACS Symp. Ser. 1997, 664, 2–19. (5) Barceló, J. Poschenrieder, Ch. Phytoremediation: principles and perspectives. Contrib. Sci. 2003, 2, 333–334. (6) Nedelkoska, T. V.; Doran, P. M. Characteristics of Heavy Metal Uptake by Plant Species with Potential for Phytoremediation and Phytomining. Miner. Eng. 2000, 13, 549–561. (7) Brooks, R. R.; Robinson, B. H. The potential use of hyperaccumulators and other plants for phytomining. Plants That Hyperaccumulate Heavy Metals; CAB International: Wallingford, U.K., 1998; pp 327–356. (8) Aldrich, M. V.; Ellzey, J. T.; Peralta-Videa, J. R.; Gonzalez, J. H.; Gardea-Torresdey, J. L. Lead uptake and the effects of EDTA on lead-tissue concentrations in the desert species mesquite (Prosopis spp.). Int. J. Phytorem. 2004, 6, 195–207. (9) Shen, Z. G.; Liu, Y. L. Progress in the study on the plants that hyperaccumulate heavy metal. Plant Physiol. Commun. 1998, 34, 133–139. (10) Baker, A. J. M.; Brooks, R. R. Terrestrial higher plants which hyperaccumulate metallic elements––a review of their distribution, ecology and phytochemistry. Biorecovery 1989, 1, 81–126. (11) Baker, A. J. M.; Reeves, R. D.; Hajar, A. S. M. Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J & C Presl (Brassicaceae). New Phytol. 1994, 127, 61–68. (12) Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. Phytoremediation potential of Thlaspi caerulescens and bladder campion for zinc- and cadmium contaminated soil. J. Environ. Qual. 1994, 23, 1151–1157. (13) Wei, C. Y.; Chen, T. B.; Huang, Z. C. Cretan bake (Pteris cretica L.): an Arsenicaccumulating Plant. Acta Ecol. Sin. 2002, 22, 777– 782. (14) Epstein, A. L.; Gussman, C. D.; Blaylock, M. J.; Yermiyahu, U.; Huang, J. W.; Kapulnik, Y.; Orser, C. S. EDTA and Pb-EDTA accumulation in Brassica juncea grown in Pb-amended soil. Plant Soil 1999, 208, 87–94. (15) Wierzbicka, M. Comparison of lead tolerance in Allium cepa with other plant species. Environ. Pollut. 1999, 104, 41–52. (16) Chaney, R. L.; Malik, M.; Li, Y. M.; Brown, S. L.; Brewer, E. P.; Angle, J. S.; Baker, A. J. M. Phytoremediation of soil metals. Curr. Opin. Biotechnol. 1997, 8, 279–284. (17) Jarvis, M. D.; Leung, D. W. M. Chelated lead transport in Pinus radiate: an ultrastructural study. Environ. Exp. Bot. 2002, 48, 21–32. (18) Sarret, G.; Vangronsveld, J.; Manceau, A.; Musso, M.; D’Haen, J.; Menthonnex, J. J.; Hazemann, J. L. Accumulation Forms of Zn and Pb in Phaseous vulgaris in the Presence and Absence of EDTA. Environ. Sci. Technol. 2001, 35, 2854–2859. (19) de la Rosa, G.; Peralta-Videa, J. R.; Cruz-Jimenez, G.; DuarteGardea, M.; Martinez, A.; Cano-Aguilera, I.; Sharma, N. C.; Sahi, S. V.; Gardea-Torresdey, J. L. The role of EDTA on lead uptake and translocation by tumbleweed (Salsola kali L.). Environ. Toxicol. Chem. 2007, 26, 1033–1039. (20) Peralta-Videa, J. R.; de la Rosa, G.; Gonzalez, J. H.; GardeaTorresdey, J. L. Effects of the growth stage on the heavy metal tolerance of alfalfa plants. Adv. Environ. Res. 2004, 8, 679–685. (21) Gardea-Torresdey, J. L.; Gonzalez, J. H.; Tiemann, K. J.; Rodríguez, O.; Gamez, G. Phytofiltration of hazardous cadmium, chromium, lead and zinc ions by biomass of Medicago sativa (Alfalfa). J. Hazard Mater. 1998, 57, 29–39. (22) López, M. L.; Peralta-Videa, J. R.; Benitez, T.; Gardea-Torresdey, J. L. Enhancement of lead uptake by alfalfa (Medicago sativa) using EDTA and a plant growth promoter. Chemosphere 2005, 61, 595–598. (23) Magidin, M.; Pittman, J. K.; Hirschi, K. D.; Bartel, B. ILR2, a novel gene regulating IAA conjugate sensitivity and metal transport in Arabidopsis thaliana. Plant J. 2003, 35, 523–534. (24) Peralta-Videa, J. R.; Gardea-Torresdey, J. L.; Tiemann, K. J.; Gomez, E.; Arteaga, S.; Rascon, E.; Parsons, J. G. Uptake and effects of five heavy metals on seed germination and plant VOL. 41, NO. 23, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

8169

(25) (26)

(27) (28)

(29)

(30) (31) (32) (33) (34) (35)

8170

growth in alfalfa (Medicago sativa L.). Bull. Environ. Contam. Toxicol. 2001, 66, 727–734. Ressler, T. WinXAS: a new software package not only for the analysis of energy-dispersive XAS data. J. Phys. IV 1997, 7(C2, X-Ray Absorption Fine Structure, Vol. 1), 269–270. Parsons, J. G.; Aldrich, M. V.; Gardea-Torresdey, J. L. Environmental and biological applications of extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) spectroscopies. Appl. Spectrosc. Rev. 2002, 37, 187– 222. Tanimoto, E. Regulation of Root by Plant Hormones––Roles for Auxin and Gibberellin. Crit. Rev. Plant Sci. 2005, 24, 249–265. Crozier, A.; Kayima, Y.; Bishop, G.; Yokota, T. Biosynthesis of hormones and elicitor molecules. In Buchanan, B. B., Gruissem,W., Jones, R. L., Eds.; Biochemistry and Molecular Biology of Plants; American Society of Plant Physiologists: Rockville, MD, 2005; 850, p 929. Spiro, M. D.; Bowers, J. F.; Cosgrove, D. J. A Comparison of Oligogalacturonide- and Auxin-Induced Extracellular Alkalinization and Growth Responses in Roots of Intact Cucumber Seedlings. Plant Physiol. 2002, 130, 895–903. Devlin, R. D. Plant Physiology, 3rd ed.; van Nostrand: New York, 1975; p 600. Hernandez-Allica, J.; Garbisu, C.; Barrutia, O.; Becerril, J. M. EDTA-induced heavy metal accumulation and phytotoxicity in cardoon plants. Environ. Exp. Bot. 2007, 60, 26–32. Hovsepyan, A.; Greipsson, S. EDTA-enhanced Phytoremediation of lead-contaminated soil by corn. J. Plant Nutr. 2005, 28, 2037– 2048. Olivares, E.,Ph.D. Dissertation, NMSU, Las Cruces, NM, 1987. Das, N. K.; Patau, K.; Skoog, F. Initiation of mitosis and cell division by kinetin and indoleacetic acid in excised tobacco pith tissue. Physiol. Plant 1956, 9, 640. Wenger, K.; Tandy, S.; Nowack, B. Effect of chelating agents on trace metal speciation and bioavailability. In Biogeochemistry

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 23, 2007

(36) (37) (38)

(39)

(40)

(41)

(42) (43)

of Chelating Agents; Nowack, B., Van Briesen, J. M., Eds.; ACS Symposium Series 910; American Chemical Society: Washington, DC, 2005; pp 204–224. Tanton, T. W.; Crowdy, S. H. Water pathways in higher plants. III. The transpiration stream within leaves. J. Exp. Bot. 1972, 23, 619–625. Sayed, S. A. Effects of lead and kinetin on the growth, and some physiological components of safflower. Plant Growth Regul. 1999, 29, 167–174. Altamura, M. M.; D’Angeli, S.; Capitani, F. The protein of RolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological stages. J. Exp. Bot. 1998, 49, 1139–1146. Romanov, G. A.; Aksenova, N. P.; Konstantinova, T. N.; Golyanovskaya, S. A.; Kossmann, J.; Willmitzer, L. Effect of indole– 3-acetic acid and kinetin on tuberisation parameters of different cultivars and transgenic lines of potato in vitro. Plant Growth Regul. 2000, 32, 245–251. Marmiroli, M.; Antonioli, G.; Maestri, E.; Marmiroli, N. Evidence of the involvement of plant ligno-cellulosic structure in the sequestration of Pb: an X-ray spectroscopy-based analysis. Environ. Pollut. (Amsterdam, The Netherlands) 2005, 134, 217– 227. Varga, A.; Zaray, G.; Fodor, F.; Cseh, E. Study of interaction of iron and lead during their uptake process in wheat roots by total-reflection x-ray fluorescence spectrometry. Spectrochim. Acta B 1997, 52B, 1027–1032. Sharma, N. C.; Gardea-Torresdey, J. L.; Parsons, J.; Sahi, S. V. Chemical speciation and cellular deposition of lead in Sesbania drummondii. Environ. Sci. Technol. 2004, 23, 2068–2073. Donda, A. F.; Giuliani, A. M. Investigation of electrode reactions of some lead complexes by means of the galvanostatic method. Ric. Sci. Rend. 1963, 3 (Sez. A; 6), 819–836.

ES0714080