Physiology and Biochemistry of Lead Accumulation in Sesbania Drummondii
This thesis presents the results of three different studies conducted to elucidate the physiology and biochemistry of lead tolerance of Sesbania drummondii. These studies were conducted to determine: 1) the presence of an antioxidant mechanism in S. drummondii to counteract oxidative stress resulting from Pb exposure, 2) the extent of Pb tolerance of S. drummondii seedlings grown in the presence of Pb and synthetic chelators in soil and 3) the expression of antioxidant enzymes in S. drummondii seedlings exposed to Pb and synthetic chelators in aqueous media. In the first study, Sesbania drummondii plants were grown in soil containing 0-10 g/kg Pb(N03)2 for 0-4 weeks, and were assessed for photosynthetic activity, growth, and Pb accumulation at each harvest. It was observed that S. drummondii seedlings were able to tolerate up to 10 g/kg Pb in soil with the only visible symptom of Pb toxicity being stunted growth. Photosynthetic efficiency of these plants remained unaffected. Plants were also grown in soil containing 7.5 g/kg Pb(N03)2in combination with chelators such as EDTA, DTPA, HEDTA, NTA and citric acid at concentrations of 0, 1.25, 2.5, 5 and 10 mmol/kg. Plants suffered little or no effect on growth and photosynthetic activity. The greatest amount of plant growth was displayed by plants grown in the presence of 7.5 g/kg Pb(N03)2and 10 mmol/kg citric acid. Accumulation of Pb in roots and shoots was also determined. This study provides evidence for the efficacy of chelate application in combination with a large-biomass Pb accumulator plant in a phytoextraction strategy. In the second set of experiments, seedlings of Sesbania drummondii were exposed to 500 mg/L Pb(NOs)2 in presence and absence of EDTA, DTPA and HEDTA for 7-28 days. Plants were assayed for metal-induced stress, which was indicated by increased activities of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD). Growth in the presence of Pb resulted in a significant elevation in activities of APX, GPX, CAT and SOD, as well as glutathione (GSH) content. However, growth in the presence of chelators (with or without Pb) resulted in a decrease in activites of APX, GPX, CAT and SOD, as well as GSH content. Chlorophyll fluorescence kinetics was also assessed by determination of Fv/Fm and Fv/F0. Exposure to Pb alone and Pb + EDTA, Pb + DTPA, and Pb + HEDTA did not affect photosynthetic integrity (Fv/F0) and efficiency (Fv/Fm). However, plant growth was affected in presence of Pb. The results suggest that Sesbania plants were able to tolerate metal stress using an antioxidant defense mechanism and therefore maintain Photosystem II activity, required for plant metabolism and physiological functions. In the third set of experiments, changes in expression of antioxidant enzymes were investigated using several common protein expression assays. Sesbania drummondii seedlings were grown in the presence and absence of Pb and synthetic chelators (EDTA, DTPA, and HEDTA). Expression of the antioxidant enzymes APX and SOD was determined using Sesbania drummondii extracts obtained by homogenization in phosphate buffer. Expression of ascorbate peroxidase was monitored by means of native gel electrophoresis. Expression of proteins in plant cells was assessed by means of SDSPAGE. Western blotting was used to compare expression of MnSOD in plants grown in the presence and absence of Pb(NC>3)2. Although Western blotting was inconclusive due to the nonspecific binding of the antibody, SDS-PAGE stained with silver stain yielded a set of bands at approximately 30kDa in which plants exposed to Pb demonstrated greater expression of this protein, which may be one or more isoforms of SOD. Together, these three studies indicate that Sesbania drummondii may have potential in use for Pb phytoextraction of contaminated sites.
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