Evaluation of the effect of EDTA and plant growth promoters on lead uptake, translocation, and oxidative stress response in Medicago sativa

Lead has been released in excess into the environment since the industrial revolution. Recently, scientists and engineers have realized that chemical compounds such as ethylenediaminetetraacetic acid (EDTA) facilitate metal uptake and translocation in certain plants. The aim of this research was to examine whether alfalfa (Medicago sativa L.), a non-lead hyperaccumulator plant species could increase its phytoextraction capability via the use of compounds like EDTA and plant hormones. This research involves the study of the combined effects of EDTA and the phytohormones indole-3-acetic acid (IAA), gibberellic acid (GA) and kinetin (KN) on lead uptake. Alfalfa plants were grown for 15 days in hydroponics under controlled conditions of pH, temperature and photoperiod. The growing media contained 40 mg Pb kg-1 [from Pb(NO3)2], without and with equimolar concentrations of EDTA (0.2 mM), and 1, 10 and 100 µM of the phytohormones IAA, GA, KN, and a mixture of IAA and KN at 100 µM each. Control plants were established for each treatment. The metal quantification in plant samples was performed by inductively coupled plasma/optical emission spectrometry and determined per kg of dry tissue (DW). The addition of EDTA/IAA (0.2 mM/100 µM) in Pb treatment significantly increased the Pb accumulation in alfalfa leaves to 2670 mg/kg DW compared to the Pb accumulation of 93 mg/kg DW in leaves of plants exposed to Pb alone and 400 mg/kg DW in plants exposed to Pb/EDTA. Gibberellic acid increased the Pb concentration in alfalfa leaves to 220 mg/kg DW compared with the Pb concentration of 30 mg/kg DW in plants treated with Pb alone. Plants treated with Pb/EDTA plus KN at 1, 10, and 100 µM increased the Pb concentrations in leaves to 910, 2340, and 3650 mg/kg DW, respectively. The increase in Pb concentration respect to the Pb/EDTA treatment was about 190, 660, and 1080%, respectively. Leaves of plants exposed to Pb/EDTA/IAA-KN at 100 µM had approximately 9,500 mg Pb kg -1 dry DW, demonstrating that hydroponically grown non-Pb hyperaccumulating plants could hyperaccumulate Pb when treated with EDTA and the mixture of IAA-KN at 100 µM each. The toxicity of lead in alfalfa plants treated with EDTA and the phytohormones was studied by catalase (CAT), ascorbate peroxidase (APOX), and total amylase (TAA) activities. The results demonstrated that CAT was significantly reduced by all treatments containing Pb, IAA, and GA at 10 and 100 µM. Treatments Pb/EDTA/KN at 1, 10, and 100 µM reduced the APOX. The TAA in leaves of alfalfa plants was significantly increased by all treatments. CAT tests showed no lead toxicity to the alfalfa seedlings. However, IAA at 10 and 100 µM revealed toxicity to the CAT enzyme. APOX tests exhibited no toxicity to the peroxidase enzyme with exception of Pb/EDTA/KN treatments. TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase enzyme in alfalfa seedlings. The concentration of Pb in roots of alfalfa plants exposed to Pb contaminated soil was 78 mg Pb kg-1 DW. In addition, when EDTA/IAA-KN was added to the soil, 142 mg Pb kg-1 DW were accumulated. The Pb concentration in alfalfa leaves when EDTA was added to the soil was 92 mg kg-1 DW and the treatment EDTA/IAA-KN showed to increase the Pb concentration in leaves to 127 mg kg-1 DW. The combination of EDTA/IAA-KN produced a synergistic effect increasing both Pb uptake and translocation. The treatments differentially affected the absorption and transport of nutritional elements. Pb reduced the absorption of K, and S; however, EDTA seems to alleviate the negative effects of Pb on the absorption of these elements. On the other hand, IAA increased the concentration of K in roots and the concentration of S in leaves, while reduced the concentration of Mg in roots. In addition, most of the treatments containing Pb reduced the concentration of microelements at root level. IAA increased the concentration of B in leaves and the concentration of Fe in roots; while reduced the concentration of Mn in root. On the other hand, the concentration of Mn in leaves was higher in plants treated with IAA at 10 µM. X-ray absorption spectroscopic studies demonstrated that Pb(II) was absorbed and translocated from roots to leaves without any change in oxidation state. The results of this investigation demonstrated that the uptake and translocation of Pb can be significantly increased by the use of EDTA and phytohormones such as IAA and KN. (Abstract shortened by UMI.