Effect of pH and EDTA on Pb Accumulation in Zea mays Seedlings

MATERIALS AND METHODS

Seeds of Zea mays Cv. single cross 704 were obtained from Agricultural Research Center of Urmia. They were cleaned, selected by the size, washed with water and detergent and finally with distilled water three times and incubated in 25°C to germinate. Four day-old seedlings with the same size were selected again and nine seedlings (as a replicate) were transferred to a plastic glass containing 50 mL of 0.25, 0.5, 1 mM Pb(NO₃)₂ with and without 0.5 mM EDTA adjusted to pH 4 and 6. After 72 h growing in an aerated (with air pump) and controlled condition (light density 16000 lux and day/night temperature 24/18°C and humidity 60%), root and shoot of seedlings were harvested separately. To show the effect of Pb on plant growth, effect of pH and EDTA on Pb toxicity, the length, fresh weight and dry weight of the roots and shoots were determined.

Pb accumulation in root and shoot: Four day old seedlings were selected and exposed to different concentrations of Pb and 0.5 mM EDTA at pH 4 and 6 as mentioned before. After 72 h root and shoot of seedlings from each of three replicate (nine seedlings per replicate) were harvested separately. Pb accumulation was determined by the method of Vassil et al.^[17] with some modification and oven dried for three days at 70°C. They were ground to a powder. 0.1 g dry matter of each replicate was digested with 5 mL concentrated HNO₃ at 180°C for 1.5 h. Then cooled at room temperature. One milliliter of 30% H₂O₂ was added to the samples and heated 180°C for 20 min. They were cooled again. Dionized water was added to the final volume of 50 mL. Pb concentration was determined using Atomic Absorption Spectrophotometer (model: 6300 Shimadzu Japan).

The effect of EDTA on Pb accumulation in shoots: Four day-old seedlings were exposed to 0.25, 0.5, 1, 1.5 and 2 mM EDTA with 0.5 mM Pb(NO₃)₂ or only 0.5 mM Pb(NO₃)₂ adjusted to pH 4. After 72 h shoots of seedlings were harvested and their Pb content was determined as described before^[17].

Effect of EDTA on water content of shoot: Water content was calculated by subtracting dry weight from fresh weight of the shoots of seedlings for each of three replicates (nine seedlings in each replicate) of different concentrations.

EDTA accumulation in shoot: Four day-old Zea mays seedlings were exposed to 0.25, 0.5, 1, 1.5 and 2 mM EDTA and 0.5 mM Pb(NO₃)₂ or only 0.5 mM Pb(NO₃)₂ adjusted to pH 4. After 72 h shoot of seedlings were harvested, frozen at -80°C. Then these materials homogenized (Heidolph Diax 900) and extracted with 1 mM of 50%(V/V) methanol, heated at 80°C for 10 min, then centrifuged at 6000 rpm for 20 min at room temperature and the supernatant was removed. The extraction process was repeated 3 times with the remaining pellet. Supernatants from all 3 extractions were pooled, dried in a speed vacuum (Heidolph LABOROTA 4003, Germany) and resuspended in 0.5 mL dionized water. These samples were then analyzed by HPLC for EDTA.

HPLC analysis of EDTA: Dried 50% methanol shoot extracts dissolved in 0.5 mL iron (III) chloride 7 mM in 0.01 M acetic acid and 0.5 mL dionized water, centrifuged at 10000 rpm for 5 min and filtered through 0.45 μm filter. HPLC analysis was performed by injection of 20 μL of sample and chromatographed on RP-C₁₈ column (30 cm, 4 mm ID., 5 μm P.S KNAUER). The column was run isocratically at 0.5 mL min^-1 with 10% methanol, 8 mM tetrabutyl ammonium hydroxide and 1mM Fe (III) chloride in 50 mM Sodium acetate/acetic acid buffer (pH 4). EDTA was detected at 254 nm using UV-vis. detector (K-2501, KNAUER) according to the method of Bergers and Degroot^[18], with some modifications.

Statistical analysis: Data analysis was done using Analysis of Variance Method (ANOVA; proc. GLM. SOSS) and ANOVA, comparing the mean of data by Dankun test and the values indicate the mean of three replicates±SE^[19].

RESULTS

We considered the effect of different concentrations of Pb(NO₃)₂ with and without 0.5 mM EDTA on Zea mays seedlings growth in pH 4 and 6. The results showed that:

Pb toxicity inhibited root elongation and biomass significantly (Fig. 1 and 2). Inhibition effect of Pb increased according to Pb concentration in growth solution (Fig. 1 and 2).

Fig. 1:	The effect of different concentrations of pb(NO3)2 with and without 0.5 mM EDTA at pH,4 and 6 on root growth of Zea mays seedlings during 72 h. (Values represent the mean±SE of three replicates)

Fig. 2:	The effect of different concentrations of pb(NO3)2 with and without 0.5 mM EDTA on dry weight of roots of Zea mays seedlings at pH 4 and 6 during 72 h. (Values represent the mean±SE of three replicates)

Fig. 3:	Pb accumulation in roots of Zea mays seedlings exposed to different concentrations of pb(NO3)2 with and without 0.5 mM EDTA at pH 4 and 6 for 72 h. (Values represent the mean±SE of three replicates)

Pb accumulation in root of seedlings in pH 4 was higher than pH 6 (Fig. 3) and the effect of low pH on Pb accumulation in root of plants was greater than EDTA (Fig. 3). As Pb content in shoot of seedlings increased by EDTA (Fig. 4), to know how much EDTA is necessary to accumulate maximum Pb in shoot of plants and what its effect is, we considered specially.

Fig. 4:	Pb accumulation in shoot of Zea mays seedlings exposed to different concentrations of pb(NO3)2 with 0.5 mM EDTA at pH4 and 6 during 72 h. (Values represent the mean±SE of three replicates)

Fig. 5:	Pb accumulation in shoots of Zea mays seedlings exposed to 0.5 mM pb (NO3)2 and also 0.5 mM pb(NO3)2 with different concentrations of EDTA at pH 4 during 72 h. (Values represent the mean±SE of three replicates)

The effect of EDTA in Pb accumulation in shoots of seedlings: Treatment of seedlings with different concentrations of EDTA with 0.5 mM Pb(NO₃)₂ showed that EDTA enhanced Pb accumulation in shoots (Fig. 5). but the highest level of Pb was at 0.5 mM EDTA with 0.5 mM Pb(NO₃)₂ in growth medium without any apparent metal toxicity effects.

Fig. 6:	The effect of only 0.5 mM pb(NO3)2 or 0.5 mM pb(NO3)2 with different concentrations of EDTA on water content of shoots of Zea mays seedlings at pH 4 during 72 h. (Values represent the mean±SE of three replicates)

Fig. 7:	A. the chromatograph of standard solution of EDTA and B: chromatograph of total EDTA of shoot of Zea mays seedlings

Effect of EDTA on water content of shoot: Treatment of Zea mays seedlings with different concentrations of EDTA with 0.5 mM Pb(NO₃)₂ decreased water content of shoot tissues in the concentrations greater than 0.5 mM EDTA in culture (Fig. 6). But water loss from the shoot was the least in equimolar concentration of Pb and EDTA (0.5 mM Pb with 0.5 mM EDTA) in medium.

EDTA accumulation in shoots: Analyzing the amount of EDTA in shoots of seedlings by HPLC (Fig. 7) indicated that the level of total EDTA increased from 0.25 mM EDTA in growth solution, reached to maximum in 1.5 mM, but its amount decreased at the concentrations more than 1.5 mM (Fig. 8).

DISCUSSION

Treatment of Zea mays seedlings with different concentrations of Pb inhibited root growth and biomass (Fig. 1 and 2); As, has been reported in rice by Yell Yang^[20]. It is because of Pb accumulation in root and its low mobility in plants^[21]. Feng^[22] implies that heavy metals decrease viscosity and elasticity of cell wall and inhibit root growth. Considering the effect of pH indicated that root elongation in pH 4 was inhibited greater than pH 6 (Fig. 1). It is due to increased Pb solubility in acidic pH and elevated availability by plant. This confirms the reports which describe that Pb solubility increases in pH ≤4^[8,23]. EDTA as a synthetic chelate makes a soluble complex with heavy metals including Pb and increases pb accumulation in shoots^[17]. For many years it has been used to supply micronutrients for plants but recently it is used to enhance heavy metal accumulation in shoots of accumulators (Indian mustard) and phytoextraction^[14]. In this experiment EDTA has also enhanced Pb accumulation in shoot of Zea mays, as a Pb-tolerant plant (Fig. 4 and 5). The results of this research indicated >2 mg kg^-1 dry weight Pb accumulated in root of Zea mays seedlings (Fig. 3) and 500 μM Pb treatment inhibited root elongation 50% (Fig. 1) while 10 μM Pb inhibited root elongation of Pb-sensitive varieties of rice 80%^[20]; but the effect of EDTA in enhancing Pb accumulation in shoots of Zea mays was not as much as Indian mustard (an accumulator)^[24]. To understand whether EDTA is absorbed by plant or not, we determined total amount of EDTA in shoots of Zea mays seedlings. The results showed that its amount in shoot of seedlings has grown up according to its concentration in growth solution. Shoot EDTA accumulation however does not always appear to be related to physiological stress; at a low EDTA concentration and neutral pH (pH 6) nearly 0.1±0.02 mM EDTA kg^-1 dry weigh was observed in shoots of Indian mustard during 48 h^[25].

Fig. 8:	EDTA accumulation in shoots of Zea mays seedlings exposed to different concentrations of EDTA with 0.5 mM pb(NO3)2 at pH 4 during 72 h. (Values represent the mean±SE of three replicates)

This suggest that, it was taken up by plants but its actual mechanism is not clearly understood. To know how EDTA influences the plants we determined water content of shoots. The results showed that (Fig. 6) EDTA caused reduction in shoot water content at the concentrations more than 0.5 mM EDTA in medium. But there was not any apparent toxicity effect on shoots at the equimolar Pb and EDTA (0.5 mM Pb(No₃)₂ with 0.5 mM EDTA). while Pb concentration in shoot was maximum. Figure 8 showed that maximum level of EDTA in shoot of plants was at treatment of 1.5 mM EDTA and this shows that EDTA is absorbed by plants and accumulated in the form of Pb-EDTA and protonated form. So to reduce toxicity effects of EDTA in plants and decrease water content loss we suggest that the rate of EDTA should be minimized or adjusted to heavy metal concentration of growth solution. We conclude from the results of this research: a) Pb is a toxic heavy metal that inhibits plant root elongation, b) Zea mays is a Pb-tolerant plant, c) pH is a major factor that influences Pb absorption by plants, d) EDTA enhances Pb accumulation in plants but it is a toxic material and causes water loss and probably some other damages to plants. So it can be used to phytoextraction not only by accumulators but also by tolerant plants. If it is used to supply plant micronutrients, determining the necessary amount and its effects on plants need further investigation^[26].

ACKNOWLEDGMENTS

We thank Dr. Abbas Samadi and Mr. Behnam Dolati for their help with atomic absorption and Dr. Naser Agh and Mr. Naser, Maleki for their help in HPLC Analysis and Mrs. Rahele Sattari for her practical assistance.