Lead Toxicity in Alfalfa Plants Exposed to Phytohormones and Ethylenediaminetetraacetic Acid Monitored by Peroxidase, Catalase, and Amylase Activities

Environmental Toxicology and Chemistry, Vol. 26, No. 12, pp. 2717–2723, 2007 䉷 2007 SETAC Printed in the USA 0730-7268/07 $12.00 ⫹ .00

LEAD TOXICITY IN ALFALFA PLANTS EXPOSED TO PHYTOHORMONES AND ETHYLENEDIAMINETETRAACETIC ACID MONITORED BY PEROXIDASE, CATALASE, AND AMYLASE ACTIVITIES MARTHA L. LO´PEZ,† JOSE R. PERALTA-VIDEA,‡ HIRAM CASTILLO-MICHEL,† ALEJANDRO MARTINEZ-MARTINEZ,§ MARIA DUARTE-GARDEA,㛳 and JORGE L. GARDEA-TORRESDEY*†‡ †Environmental Science and Engineering Ph.D. Program, The University of Texas at El Paso, El Paso, Texas 79968, USA ‡Chemistry Department, The University of Texas at El Paso, El Paso, Texas 79968, USA §Instituto de Ciencias Biome´dicas, Universidad Auto´noma de Ciudad Jua´rez, Ciudad Jua´rez, Chih., Me´xico, C.P. 32310, Mexico 㛳Department of Health Promotion, College of Health Science, The University of Texas at El Paso, El Paso, Texas 79968, USA ( Received 14 May 2007; Accepted 24 July 2007) Abstract—This manuscript describes the toxicity of lead in alfalfa plants treated with ethylenediaminetetraacetic acid (EDTA) and the phytohormones indole-3-acetic-acid (IAA), gibberellic acid (GA), and kinetin (KN), on catalase (CAT), ascorbate peroxidase (APOX), and total amylase activity (TAA). In all cases Pb was used at 40 mg/L; EDTA at 0.2 mM (equimolar to Pb); and IAA, GA, and KN at 1, 10, and 100 ␮M, respectively. An experiment containing Pb at 40 mg/L, 0.2 mM EDTA, and IAA and KN at 100 ␮M each was performed to determine changes in TAA. A control (plain nutrient solution) also was used for comparison. In all cases the treatments were performed in triplicate. Standard procedures were followed to determine the activity of the respective enzymes. After 10 d of exposure to the treatments, the leaves were harvested, homogenized, and centrifuged, and the supernatants were analyzed for CAT, APOX, and TAA. All determinations were performed in triplicate. The results demonstrated that CAT was reduced significantly ( p ⬍ 0.05) by all treatments containing Pb, IAA, and GA at 10 and 100 ␮M. However, only the treatments Pb/EDTA/KN at 1, 10, and 100 ␮M reduced the APOX. The TAA in leaves of alfalfa plants was increased significantly ( p ⬍ 0.05) by all treatments. Overall, the results suggest that the CAT tests showed no lead toxicity to the alfalfa seedlings. However IAA at 10 and 100 ␮M revealed toxicity to the CAT enzyme. In addition, the APOX tests exhibited no toxicity to the peroxidase enzyme with the exception of Pb/EDTA/KN treatments. Finally, the TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase enzyme in alfalfa seedlings. Keywords—Amylase

Catalase

Lead

Peroxidase

Phytohormones

compounds. However, excess of ROS can produce breakdown of proteins, lipid peroxidation in membranes, and DNA injury [7]. Stress induced by heavy metals and other factors can be quantified by the specific activity of antioxidant enzymes such as catalase (CAT 1.11.1.6) and ascorbate peroxidase (APOX 1.11.1.11), which are able to remove the excess of additional ROS molecules [5,8]. Amylase is another enzyme that can be used to study the plant–metal interactions. Amylase (Enzyme Commission 3.2.1) is a starch-degrading enzyme that catalyzes the hydrolysis of starch and some other polysaccharides in bacteria, animals, and plants [9]. Plant health can be determined based on the content of metabolites as well as amylase activity [10]. Several researchers have studied the storage and starch metabolism in Medicago sativa because the roots of this model plant have a high starch content and amylase activity [11]. It has been determined that phytohormones are naturally occurring substances often present at the microgram level within plants [12]. Indole-3-acetic acid (IAA), gibberellic acid (GA), and kinetin (KN) are examples of phytohormones involved in different developmental processes such as cell division and elongation [13]. Indole-3-acetic acid is a molecule that promotes apical dominance, tropism, stem elongation, and root formation among others. Gibberellic acid belongs to the tetracyclic diterpenes group that is related to germination and flowering processes, and KN upholds cell division, delays senescence of leaves, and influences the opening of stomata [14]. In previous studies, alfalfa plants were exposed hydropon-

INTRODUCTION

Plants exposed to stressing agents such as drought, salinity, excess of heavy metals, air pollutants, or pathogens have developed strategic defense mechanisms that vary between species and the nature of stressing agent [1]. Lead is a heavy metal with no known function in living systems, which has proven to be toxic for plants and animals [2]. Lead usually enters plants by similar pathways as micro- and macronutrients. However, the metabolic pathways underwent by this element within plant cells are not fully understood [3]. Studies have shown that ethylenediaminetetraacetic acid (EDTA) improves the translocation of Pb from roots to leaves in plants cultured in hydroponics [4]. The translocation of Pb at threshold levels induces intracellular injuries and alters metabolic processes affecting plant growth [5]. Due to that reason, researchers are challenging both the uptake and translocation of Pb and other metals to improve phytoremediation techniques without severe tissue damage. The absorption of heavy metals such as Pb induces the formation of reactive oxygen species (ROS) in plant cell organelles such as peroxisomes and chloroplasts [6]. Reactive oxygen species such as hydrogen peroxide, hydroxyl, and superoxide radicals are byproducts originated in plants from reactions such as photosynthesis and other metabolic pathways. Plants have special mechanisms to remove or inactivate these * To whom correspondence may be addressed ([email protected]). Published on the Web 8/06/2007. 2717

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ically to Pb, EDTA, and several concentrations of IAA. Plants exposed to 40 mg Pb L⫺1 and treated with IAA/EDTA (100 ␮M/0.2 mM) increased their Pb concentration in leaves by about 2,800% as compared to the Pb concentration in leaves of plants exposed to Pb alone [15]. In addition, GA and KN also were tested in alfalfa for improving the Pb uptake in hydroponics. The results have shown that the leaves of alfalfa plants exposed to 40 mg Pb L⫺1 plus KN/EDTA (100 ␮M/0.2 mM) had 2,275% more Pb than the leaves of plants exposed to the Pb/EDTA treatment (data not shown). The present manuscript describes the effects of Pb, IAA, GA, KN, and EDTA on CAT, APOX, and total amylase activity (TAA) in alfalfa plants grown in hydroponics. Standard procedures were followed to determine the activity of the respective enzymes.

M.L. Lo´pez et al.

Determination of APOX The ascorbate peroxidase activity was evaluated according to Murgia et al. [17] with minor modifications. Extracts of leaves were prepared as described above. The supernatant was transferred to a microtube and assayed after centrifugation. A volume of 886 ␮l of 0.1 M KH2PO4 buffer at pH 7.4, 10 ␮l of the 17 mM H2O2, 100 ␮l of the sample, and 4 ␮l of a 25 mM solution of ascorbate were placed in a quartz cuvette (final volume, 1 ml) and mixed by hand shaking (three times). The absorbance was recorded at 265 nm in a PerkinElmer Lambda 14 ultraviolet/vis spectrometer. The absorbance was recorded as described above and the extinction coefficient for H2O2 was set experimentally at 19.19 mM⫺1 cm ⫺1.

Determination TAA MATERIALS AND METHODS

Seeds of alfalfa (Medicago sativa L.) were acquired from the California Crop Improvement Association ([CCIA], Davis, CA, USA). The seeds were treated as previously described [15]. Briefly, the seeds were soaked for 5 min in 100% ethanol, washed with sterilized deionized water, exposed to a 4% sodium hypochlorite solution for 30 min while stirring, washed with deionized water, and placed in sterilized paper towels dampened with an antibiotic-antimycotic solution (Sigma A5955, St. Louis, MO, USA) to avoid fungal and bacterial contamination. After 4 d in the dark, the seedlings were exposed to light for 1 d and transferred to 250-ml jars containing a low phosphate Hoagland nutrient solution amended with Pb, EDTA, and 1, 10, or 100 ␮M of the hormones IAA, GA, and KN. The following 30 treatments were set: Control, 1 ␮M hormone, 10 ␮M hormone, 100 ␮M hormone, Pb, Pb/1 ␮M hormone, Pb/10 ␮M hormone, Pb/100 ␮M hormone, Pb/ EDTA, Pb/EDTA/1 ␮M hormone, Pb/EDTA/10 ␮M hormone, Pb/EDTA/100 ␮M hormone. Each solution was adjusted to pH 5.4 using 0.1 M NaOH and 0.1 M HCl as required. In all treatments the Pb concentration was kept at 40 mg L⫺1 and EDTA at 0.2 mM (equimolar to Pb). Three replicates/treatment were placed in 250-ml jars and set under a 12-h light:dark cycle and illumination of 45 mmol m⫺2 s⫺1. After 10 d of exposure, the leaves were harvested, homogenized, centrifuged, and the supernatants were analyzed for CAT, APOX, and TAA. All determinations were performed in triplicate.

Determination of CAT The activity of catalase in leaves of alfalfa plants was determined according to Gallego et al. [16] with minor variations. For each treatment, a ratio of 10% weight:volume of leaf sample was homogenized with phosphate buffer (0.100 g of leaves mixed with 900 ␮l of 25 mM KH2PO4 at pH 7.4). The mixture was centrifuged for 5 min at 4,500 rpm on a bench centrifuge (Fisher Scientific Model 8K, Pittsburgh, PA, USA). The supernatant was transferred to a micro tube for the assay. A sample of 990 ␮l of H2O2 was placed in a quartz cuvette and 10 ␮l of the sample were added to obtain a final volume of 1 ml. This was hand shaken (three times) to mix, and the absorbance at 240 nm was recorded in a Perkin Elmer Lambda 14 ultraviolet/visible spectrometer (single-beam mode, Perkin Elmer, Uberlinger, Germany). The absorbance values were obtained from the first linear section of slopes between 0.5 and 1 min. The extinction coefficient for H2O2 was set at 19.19 mM⫺1 cm ⫺1. A standard of bovine serum albumin and protein content from the extracted sample were used in this research.

The total amylolitic activity was assayed according to the Fuwa colorimetric method, an iodine-starch color reaction [18]. The assay was modified to fit a microplate format (H. Castillo-Michel, 2005, Master’s thesis, University of Texas at El Paso, TX, USA). Briefly, the enzyme extracts were prepared from fresh plant tissues at 10% in imidazole buffer (0.2 g in 1.8, in 2 mM imidazole, pH 7.0) by using a glass–glass homogenizer. Extracts were centrifuged for 5 min at 4⬚C and 13,000 rpm in a refrigerated centrifuge (Eppendorf AG bench centrifuge 5417 R, Hamburg, Germany). A reaction mixture containing 400 ␮l of the enzyme extract and 700 ␮l of 1% starch solution in 2 mM imidazole buffer (pH 7.0) was prepared in Eppendorf tubes. The reaction was stopped by adding an aliquot of 150 ␮l from the reaction mixture to 200 ␮l of cold trichloroacetic acid at 0, 20, 40, 60, 80, and 90 min. When the reaction was stopped, an aliquot of 30 ␮l was mixed with 300 ␮l of iodine reagent (0.0075% iodine and 0.075% potassium iodide) previously added to the microplate wells. The absorbance at 660 nm was measured in a microplate reader at room temperature (Pierce, Rockford, IL, USA) approximately 30 min after iodine-starch blue color was developed. The specific activity was calculated taking into account the molar extinction coefficient for starch and dilution factors, as well as fresh weight. The molar extinction coefficient average for starch in the treatments was 0.54889 ⫾ 0.0403 % abs⫺1 path⫺1.

Statistics The values reported are the average of three replicates ⫾ standard error obtained from the one-way analysis of variance followed by Tukey’s honestly significant difference analysis performed with the statistical package SPSS Version 12.0 (SPSS, Chicago, IL, USA). When stated, t tests and Pearson correlation coefficients were also used. In all cases the statistical significance is based on a probability of p ⬍ 0.05, unless otherwise stated. RESULTS AND DISCUSSION

Effect of IAA, GA, and KN on CAT The activity of catalase in leaves of alfalfa plants treated with IAA, Pb/IAA, and Pb/EDTA/IAA is shown in Figure 1A to C, respectively. As shown in Figure 1A, the CAT in leaves of control plants was about 170 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein. The CAT increased approximately 50% in leaves of plants exposed to 1 ␮M IAA. However, IAA at 10 ␮M and above reduced the activity of this enzyme. Furthermore, CAT in leaves of plants treated with 100 ␮M IAA was reduced significantly compared to the CAT of control

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Fig. 1. Catalase- and ascorbate peroxidase–specific activities in leaves of alfalfa plants exposed for 10 d to: (A) Indole-3-acetic-acid (IAA) at 0 (control), 1, 10, and 100 ␮M; (B) Pb, Pb/IAA at 1, 10, and 100 ␮M; and (C) Pb/ethylenediaminetetraacetic acid (EDTA), Pb/EDTA/ IAA at 1, 10, and 100 ␮M. In all cases Pb was at 40 mg/L and EDTA was 0.2 mM. Data are average of three extracts. Error bars represent standard error.

Fig. 2. Catalase- and ascorbate peroxidase–specific activities in leaves of alfalfa plants exposed for 10 d to: (A) Gibberellic acid (GA) at 0 (control), 1, 10, and 100 ␮M; (B) Pb, Pb/GA at 1, 10, and 100 ␮M; and (C) Pb/ethylenediaminetetraacetic acid (EDTA), Pb/EDTA/GA at 1, 10, and 100 ␮M. In all cases Pb was at 40 mg/L and EDTA was 0.2 mM. Data are average of three extracts. Error bars represent standard error.

leaves. Michniewicz and Stanislawski [19] found that CAT in seeds of Leszczyhska wzesha exposed to IAA (0.001%) increased during the germination period and decreased in seedlings without affecting plant growth. In the present study, CAT in plants exposed to 40 mg Pb L⫺1 increased by about 31% compared to control leaves (Fig. 1B). Similar activity was observed in leaves of plants treated with Pb/1␮M IAA. Heavy metals such as Pb increase the CAT activity because of the direct stimulation of enzyme synthesis as a result of ROS formation inside plant tissues [20]. Nevertheless, plants treated with Pb/IAA at 10 ␮M had similar CAT activity as control leaves (Fig. 1B). Likewise, plants exposed to Pb plus 100 ␮M IAA had CAT activity similar to plants exposed to IAA at 100 ␮M (Fig. 1A). These results corroborated that IAA at 10 and 100 ␮M concentrations inhibited CAT in alfalfa. According to Gazaryan and Lagrimini [21] this apparent CAT inhibition could be due to IAA, as a strong antioxidant against hydroxyl radicals, forming IAA hydroperoxide. On the other hand, Figure 1C shows that the CAT activity of plants treated with Pb/ EDTA/IAA at 1 ␮M was similar to the CAT in control leaves. This result suggests that the EDTA/Pb complex was less toxic for the plants. According to Ruley et al. [8], chelators alleviate the toxic effects caused by lead and produce different antioxidant responses in plants. Nevertheless, CAT in plants treated with Pb/EDTA/IAA at 10 and 100 ␮M was reduced by approximately 31 and 54%, respectively, compared to control

leaves. This was may be due to the fact that IAA inhibited CAT when supplied to the media at 10 and 100 ␮M concentrations. The activity of catalase in leaves of alfalfa plants treated with GA, Pb/GA, and Pb/EDTA/GA are shown in Figure 2A to C. Control plants had a CAT of 170 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein, although plants treated with GA at 1, 10, and 100 ␮M had CAT activities of 40, 110, and 70, respectively (Fig. 2A). The type of inverted response in CAT to the GA concentrations might be due to the hormesis phenomenon, a typical dose-response to stressing factors [22]. Abdel-Kader [23] found that GA applied at 50 and 100 mg L⫺1 three weeks after a drought period, reduced CAT in lettuce cultivar baladi. It has been reported that GA increases protein synthesis and alleviates the effects caused by water stress [23]. In the present study, CAT in leaves of plants exposed to Pb alone increased by approximately 15% compared to control leaves. However, in plants treated with 40 mg Pb L⫺1 and GA at 1 ␮M, CAT was reduced by about 70% compared to plants exposed to Pb alone (Fig. 2B), although the CAT in plants exposed to Pb plus GA at 10 and 100 ␮M was similar to the CAT in control leaves. The results for CAT in the present study were similar to the results found by Moya et al. [24], who reported that external GA diminished the effects caused by heavy metals in rice plants through the mobilization of carbohydrate reserves.

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Fig. 3. Catalase- and ascorbate peroxidase–specific activities in leaves of alfalfa plants exposed for 10 d to: (A) Kinetin (KN) at 0 (control), 1, 10 and 100 ␮M; (B) Pb, Pb/KN at 1, 10, and 100 ␮M; and (C) Pb/ethylenediaminetetraacetic acid (EDTA), Pb/EDTA/KN at 1, 10, and 100 ␮M. In all cases Pb was at 40 mg/L and EDTA was 0.2 mM. Data are average of three extracts. Error bars represent standard error.

Figure 2C shows that CAT in leaves of alfalfa plants exposed to Pb/EDTA/GA at 1, 10, and 100 ␮M decreased by approximately 25, 75, and 50%, respectively. This decrement in CAT in plants treated with Pb/EDTA/GA probably was due to an increase in Pb concentration in leaves, because more Pb was translocated from the roots to the leaves (data not shown). Lead can bind to sulfhydryl groups (–SH) of enzymes, disturbing their normal activity [1]. Moya et al. [24] found that the accumulation of heavy metals in rice leaves produced changes in carbohydrate and protein content, and GA at 19 ␮M amplified the toxic effects inhibiting plant growth and sugar accumulation. The CAT was inhibited especially in plants treated with Pb/EDTA/GA at 10 ␮M, which were found to have the highest Pb concentration (data not shown). The results suggest that the antioxidant enzyme activity could increase/decrease as a response of the Pb toxicity and the growth

stage of the plant. Further experiments are needed in order to study the role of GA on CAT in alfalfa leaves. The activity of catalase in leaves of alfalfa plants treated with KN, Pb/KN, and Pb/EDTA/KN are shown in Figure 3A to C. In control plants, CAT was 130 (␮mol of H2O2 decomposed min⫺1 mg⫺1 protein; Fig. 3A). No changes were observed in the CAT of plants treated with KN at 1 ␮M. However, the activity of this enzyme increased by approximately 50% in plants treated with 10 and 100 ␮M KN. These results were opposed to those obtained for IAA and GA in which CAT was reduced in plants treated with higher concentrations of IAA and GA (10 and 100 ␮M). The CAT in leaves of plants exposed to Pb alone and KN at 100 ␮M was similar (⬃30% higher compared to control leaves, Fig. 3B). Furthermore, CAT activity in leaves of plants exposed to Pb plus KN increased as the concentration of KN in the medium increased. This result

Lead toxicity in alfalfa exposed to phytohormones and EDTA

indicated an additive stress from both Pb and KN. El-Kady et al. [25] found an increase on CAT in Datura plants treated with different exogenous concentrations of KN. El-Meleigy et al. [26] found that KN sprayed on Arachis hypogaea L. (10 mg/L) increased CAT to diminish the effects of drought. According to Erdei et al. [27], the time of exposure and metal concentration in the media induce changes in antioxidant enzymes such as CAT, superoxidedismutase, APOX, and others. The addition of equimolar concentrations of EDTA to treatments containing Pb plus KN at 1 and 10 ␮M did not produce changes in CAT. However, the addition of EDTA to the treatment Pb/KN at 100 ␮M reduced CAT by approximately 50% (120 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein) compared to CAT in leaves of plants treated with Pb alone or Pb/EDTA (Fig. 3B and C). This was may be due to the fact that Pb accumulated at higher concentrations in leaf of alfalfa plants exposed to Pb/EDTA/KN at 100 ␮M (data not shown).

Effect of IAA, GA, and KN on APOX The ascorbate peroxidase activity in leaves of alfalfa plants treated with IAA, Pb/IAA, and Pb/EDTA/IAA is shown in Figure 1A to C. Figure 1A shows that control plants had an APOX of 123 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein. The APOX increased twofold (250 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein) when IAA at 1 ␮M was added to the media. However, APOX activity in plants treated with 10 and 100 ␮M IAA was 150 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein, which indicated an increase of 25% compared to control plants. Kumar and Khan [28] studied the change on enzymatic activity in leaves of ragi (Eleusine coracana Gaertin. cv PR 202) at different growth stages. These researchers found that, in leaves of this plant, phytohormones such as IAA, KN, and GA increased APOX at senescence. It has been reported that excess IAA increases the production of H2O2 and O⫺2 , which increases the production of antioxidant enzymes [29]. When alfalfa plants were exposed to Pb at 40 mg L⫺1, the APOX increased approximately15% compared to the control (Fig. 1B). This response has been observed in other plant species. Verma and Dubey [5] have shown that Pb increased APOX in roots and shoots of rice. In the present study, plants exposed to Pb/IAA at 1 ␮M had an APOX approximately 20% lower compared to plants exposed to Pb alone. However, when the medium contained Pb plus IAA at 10 and 100 ␮M, the APOX increased by approximately 33 and 46%, respectively, compared to plants treated with Pb alone. Comparing Figure 1A and B, one can see that IAA and Pb had an additive effect in the increase of APOX activity. Figure 1C shows the trend in APOX activity when EDTA and IAA were added to the growth medium. Plants exposed to Pb/EDTA showed an APOX of 280 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein, 127% higher than the activity from control leaves and 86% higher than the APOX shown by the plants exposed to Pb alone. The increase in APOX could be explained by the increase in Pb concentration in leaves of plants exposed to Pb/ EDTA (⬃300%) compared to the concentration of Pb in leaves of plants treated with Pb alone [15]. Lead/EDTA/IAA treatments showed the highest APOX (between 350 and 400 ␮mol of H2O2 decomposed min⫺1 mg⫺1 protein). Sharma and Dubey [1] reported that Pb increased APOX in leaves of soybean plants. The ascorbate peroxidase activity in leaves of alfalfa plants exposed to Pb, Pb/EDTA, and Pb/EDTA/GA are shown in Figure 2A to C. As seen in Figure 2A, none of the GA con-

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centrations significantly affected the APOX. Figure 2B to C shows that Pb and Pb/EDTA treatments increased APOX by approximately 15 and 50%, respectively, compared to control plants (Fig. 2A). However, APOX did not change significantly when GA was added at 10 and 100 ␮M. Furthermore, the combination of Pb/EDTA/GA did not change the APOX compared to the Pb/EDTA treatment. As explained before, the increase in Pb concentration in leaves of plants treated with Pb and EDTA could be responsible for the APOX increase in plant exposed to Pb/EDTA [15]. The ascorbate peroxidase activity in plants treated with KN, Pb/KN, and Pb/EDTA/KN are shown in Figure 3A to C. Figure 3A shows that none of the KN concentrations changed the APOX in alfalfa seedlings. Figure 3B shows that the APOX increased approximately 20 and 60%, compared to the control, in plants exposed to Pb and Pb/KN at 1 ␮M, respectively. Plants treated with Pb plus KN at 10 and 100 ␮M had an APOX similar to plants treated with Pb alone. However, APOX increased about 124% in plants treated with Pb/EDTA/KN at 1 ␮M, which could be explained by the increase in Pb concentration in leaves (data not shown). The addition of KN at 10 and 100 ␮M to the medium containing Pb and EDTA reduced APOX to the levels produced by Pb/KN. Probably, the plants were intoxicated by an excess of Pb translocated to the leaves, losing their defensive capacity (data not shown). According to Verma and Dubey [5], Pb increases APOX due to the production of ROS. However, Pb can be deposited in vacuoles as Pb/EDTA complex producing changes in the enzymatic activity. These changes are related to metal concentration, growth stage, and resistance to heavy metals [30]. ElMeleigy et al. [26] reported that, in plants of Arachis hypogaea L. stressed by water deficit, KN decreased the APOX.

Total amylase activity The total amylolitic activity was assayed as described by Fuwa [18], with several modifications (H. Castillo-Michel, Master’s thesis). The rate hydrolyzed-starch min⫺1 mg⫺1 fresh weight was determined at room temperature. As shown in Figure 4A, TAA in control leaves was 0.0006 ⫾ 0.0003%, the smallest value from all treatments. The addition of IAA alone increased TAA fivefold compared with control leaves (Fig. 4A). This value was reduced slightly and maintained in plants treated with Pb/EDTA/IAA (Fig. 4A), IAA/KN, Pb/IAA/KN, and Pb/EDTA/IAA/KN (Fig. 4B), suggesting that the effect of IAA was independent of the effect of Pb alone. Kadiri and Hussaini [31] reported that IAA increased alpha-amylase activity in Sorghum bicolor. In peas (Pissum sativum L.), the ␣-amylase at the fourth day of exposure to 5 mM Cd (a metal of similar redox potential as Pb) experienced an increase of nearly four times compared to control plants [32]. On the other hand, Mukherjee and Maitra [33] have reported an inhibition of protease and amylase activities by approximately 50% in rice endosperm exposed to Pb(II). The induction of IAA synthesis, as well as the enhancement of ␣-amylase activity, had been shown previously in rice [34]. In Figure 4A, it can be seen that Pb alone increased the TAA almost four times compared to the control. However, when EDTA equimolar to Pb was added to the treatment, TAA was reduced to half compared to the value found with Pb alone. This decrease in TAA probably was due to the fact that the complex EDTA-Pb is less toxic than Pb alone. On the other hand, EDTA can inhibit amylase activity [35]. The results suggest that additional concentrations of IAA and KN stimulated amylase activity in all

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Fig. 4. Total amylase activity in leaves of alfalfa plants exposed for 10 d to: (A) Control, indole-3-acetic acid (IAA), Pb, Pb/ethylenediaminetetraacetic acid (EDTA), Pb/EDTA/IAA, and (B) kinetin (KN), Pb/EDTA/KN, KN/IAA, Pb/IAA/KN, and Pb/EDTA/IAA/KN. In all cases Pb was at 40 mg/L, KN and IAA at 100 ␮M, and EDTA at 0.2 mM. Data are average of three extracts. Error bars represent standard error.

treatments. These results are similar to those found by Hirasawa [36], who reported that ␣-amylase activity increased in cotyledons of pea plants treated with IAA. Endogenous auxin concentrations promoted ␣-amylase activity because of the decomposition of IAA in pea cotyledons [36]. According to Sayed [37], plants grown in Pb-contaminated soils decreased its soluble sugars and stability of leaf membranes. The addition of KN helped to diminish these effects, more biomass was produced in shoots and, as a result, the amylase activity increased in order to metabolize the storage sugars. As shown in Figure 4A to B, the treatments containing Pb had less TAA compared to plants exposed to IAA, KN, and IAA/KN. These results suggest that IAA and KN stimulate mobilization of carbohydrates that allow growth, expansion, and maturity of plant tissues. Tang and Newton [38] reported that auxins combined with cytokinins induced cell differentiation in Pinus strobus L. In addition, Bewli and Witham [39] found that kinetin-increased amylase activity in cotyledons of beans might be due to the increase of starch hydrolysis required for development and enlargement of tissues. The results suggest that the stress produced by Pb uptake is enhanced by the addition of EDTA, IAA, and/or KN. In order to overcome the stress, plants increased the TAA. These results are similar to those found by Clifford et al. [40], who reported that glucose and starch content significantly declined in leaves of the drought-tolerant species Ziziphus mauritiana Lamk and Z. rotundifolia Lamk grown in a free-draining medium containing equal parts of Levingtons C2, grit and John Innes No. 2.

CONCLUSION

In all cases, CAT was reduced when the concentration of IAA in the medium was at 10 and 100 ␮M and in all treatments containing GA. However, CAT was increased by Pb and reduced by the addition of EDTA. In addition, all treatments containing KN, except Pb/EDTA/KN at 100 ␮M, increased CAT. All treatments containing IAA/EDTA, Pb/EDTA/GA, Pb alone, Pb/EDTA, Pb/IAA at 10 and 100 ␮M, and IAA at 1 ␮M significantly increased APOX. The highest activity was found in Pb/EDTA/IAA at 1 ␮M. Gibberellic acid and KN did not increase the APOX. In general, IAA and KN, as well as Pb and Pb/EDTA, treatments increased the TAA. Overall, the results suggest that the CAT tests showed no lead toxicity to the alfalfa seedlings. However IAA at 10 and 100 ␮M revealed toxicity to the CAT enzyme. In addition, the APOX tests exhibited no toxicity to the peroxidase enzyme with exception of Pb/EDTA/KN treatments. Finally, the TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase enzyme in alfalfa seedlings. Acknowledgement—The authors acknowledge the financial support from the National Institutes of Health (grant S06GM8012-33) and the Historic Black Colleges and Universities/Minority Institutions Environmental Technology Consortium that is funded by the U.S. Department of Energy. We also acknowledge the University of Texas at El Paso’s Center for Environmental Resource Management through funding from the U.S. Environmental Protection Agency. J. GardeaTorresdey acknowledges the Dudley family for the Endowed Research Professorship in Chemistry and the University of Texas System Library, Equipment, Repair, and Rehabilitation Program. M.L. Lopez

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