| As the in-depth study of heavy metal hyperaccumulator, how to strengthen the ability of hyperaccumulator to accumulate more heavy metal, and understand fully the mechanism of heavy metal endurance and absorbing/ accumulating was the focus of present scientific research. In these study the two ecotypes of Sedum alfredii Hance was used to as material, hydroponic experiments were conducted during present studies to investigate systemically the influence of Zn accumulating under exogenous addition of different concentrations of calcium ions, role of EDTA in alleviating lead toxicity in accumulator species of Sedum alfredii H., the lead endurance and process of antioxidative metabolize. In order to validate the detoxify function of antioxidative enzyme and antioxidants through the comparison of accumulating ecotype (AE) and non-accumulating ecotype (NAE) of S. alfredii using histochemical analyses, integrated couple plasma mass spectrophotometry (ICP-MS) and fluorescence microscopy. The main results were as follows:1. A hydroponic study was conducted on biomass, root morphology, zinc (Zn), calcium (Ca) and sulfur (S) contents in the two ecotypes of Sedum alfredii H. under exogenous addition of different concentrations of calcium ions. The results showed that the dry mass of both ecotypes of S. alfredii H. increased with increasing calcium concentration, and the shoot dry mass of hyperaccumulating ecotype increased significantly (P<0.05), the dry mass increasing of no-hyperaccumulating ecotype was not significant. The highest shoot/root ratio of hyperaccumulating ecotype was under 4 mmol L-1Ca2+, but no-hyperaccumulating ecotype was at 1 mmol L-1Ca2+, the ratio declined as the increasing of external Ca2+ level. Root length, root surface area of hyperaccumulating ecotype increased in respond to increasing calcium concentration in nutrient medium, while Ca decreased the root length and root surface area of non-hyperaccumulating ecotype. As the increasing of external Ca2+ level, Root average Diameter, root volume and lateral roots of no-hyperaccumulating ecotype has declined, while these indexs of hyperaccumulating ecotype were no obvious change.2. Zinc accumulation in root, stem and leaf of hyperaccumulating ecotype were increased by the increasing calcium concentration, but the difference among the treatments was not significant (P<0.05). However, the Zn accumulation in shoots of non-hyperaccumulating ecotype was significantly low (P<0.05).The Ca accumulation in root, stem and leaf in non-hyperaccumulating ecotype were significantly correlated with the external calcium concentration in nutrient medium (P<0.05).The S accumulation in roots of hyperaccumulating ecotype of were significantly correlated with external calcium concentrations (p<0.01). The trendes of change in Zn and S of hyperaccumulating ecotype was accordant (Y= 4.05X - 1808.29, R2=0.9153). Extracellular calcium concentration promoted the Zn absorption and accumulation in hyperaccumulating ecotype, while, the increase in calcium concentration inhibited the Zn accumulation in non-hyperaccumulating ecotype. Appropriate concentration of calcium increase and promote growth of hyperaccumulating ecotype of Sedum alfredii H., and also to improve its ability to accumulate more zinc.3. Loss of plasma membrane integrity became serious by increasing Pb concentration in the medium, 200μM Pb + 200μM EDTA has alleviated the root cell death, even there was no influence on root of alone EDTA treatment. The biomass was significantly affected by high concentration of Pb, and root growth was also affected by EDTA-Pb compared with ionic Pb. The concentration of reactive oxygen species (ROS) was determined by fluorescence microscopy, which indicates that the Pb stress increased the content of ROS significantly, whereas the EDTA-Pb decreased the burst of H2O2. High Pb concentrations increase the activity of SOD and LOX, EDTA-Pb resulted in decline the SOD and LOX activity of roots and shoots.4. Lead accumulation was higher in the samples treated with ionic lead than that of the control. The Cu concentration in root increased significantly under Pb and EDTA-Pb treatment, and 200μM Pb markedly increased the Fe content in roots. Under ionic Pb condition, the contents of Mg, Ca and K in shoots decreased, whereas they were significantly increased in case of EDTA-Pb. These results suggested that accumulating ecotype of S. alfredii roots were inefficient in uptake of higher concentration of EDTA-chelated Pb for long treatment duration, and that lead toxicity could be alleviated by EDTA. 5. This study indicates concentration and spatial positive correlations among the events of loss of integrity of the plasma membrane, lipid peroxidation and Pb accumulation investigated in two ecotypes of S. alfredii H. roots. The loss of plasma membrane integrity was engendered under 50μM Pb in AE root and 25μM Pb in NAE root, especially in the region of cell division and procambium elongation. The lipid peroxidation engendered in AE roots at 25μM and in NAE roots at 10μM. Both of the loss of plasma membrane integrity and lipid peroxidation became serious due to the increasing of Pb concentration in the medium for 24 h treatment. The two events were observed clearly at the periphery of cracks in the roots, and gradually seen inside the cracks by increasing the lead concentration. The high tolerance lead level of S. alfredii H·in Hoagland medium showed 50μM of accumulating ecotype and 25μM of no-accumulating ecotype. Exceeding the large amounts of this concentration can consider as sublethal Pb level. It is concluded that Pb accumulating ecotype of S. alfredii H. was stronger lead endurance than no-accumulating ecotype.6. Reactive oxygen species accumulation have raised as the increasing of lead level. O2·- content in roots of accumulating ecotype was lower than no-accumulating ecotype, the other way round H2O2 level in roots of accumulating ecotype was higher than no-accumulating ecotype. The difference of two ecotypes was significant after 25μM lead exposure (p<0.05). The lipid peroxidation was some higher in no-accumulating ecotype roots than accumulating ecotype. The induction of antioxidant enzymes including SOD, CAT, POD, APX, and LOX is an important protective mechanism to minimize Pb toxicity results in the oxidative damage. Also, the enhanced level of AsA and GSH suggests its active participation in detoxification of toxic oxygen species. The antioxidative metabolize system of accumulating ecotype of S. alfredii H. has instantaneous response to quench the burst of reactive oxygen species. |
