Physiological Mechanisms Of Cd Hyperaccumulation By The ASME Of Sedum Alfredii H. And Its Regulation

In the present study, the Cd uptake and endurance mechanisms of the ancient silver-mining ecotype of Sedum alfredii Hance (ASME.) were systematically studied using the mine area investigation, hydroponic and pot experiments, the established microplate-based fluorescence method, transmission electron microscopy (TEM) technique, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method. The influences of three selected antioxidant substances in the Cd endurance of ASME were also investigated. The main results obtained were as follows:1. Based on the results obtained by the ancient silver-mine area investigations and the hydroponic experiments, the Cd content in the above-ground of the ASME discovered from an ancient silver mine in Quzhou, Zhejiang Province, China, reached to 893 mg kg^-1 on field condition, and the relevant enrichment coefficients (the ratio of Cd content in plant to Cd content in the soil) was as high as 22. The ASME still grow normally even at the Cd concentration as high as 75.0 mg kg^-1. And it can endure the Cd concentration as high as 400μM and the accumulation of Cd content in the leaves of ASME reached as high as 6556 mg kg^-1 on hydroponic experiments. The results showed that the ASME was identified with the characteristics of Cd hyperaccumulation.2. In this study, a new high selective and sensitive microplate-based fluorescence detection method for the determination of nitric oxide (NO) released from plants has been developed, in which a specific newly-synthesized fluorescence indicator - DAMBO-P^H was selected as the NO probe. Various parameters affecting chemical derivatization in microplate form were systematically studied. The limits of detection (LOD) of the proposed method was 0.19 nM, whereas the LODs of the spectrophotometric and the spectrofluorimetric methods were 100-200 nM and 10-20 nM, respectively.3. The suitability of capillary chromatography (CE) with laser-induced fluorescence (LIF) detection for the determination of melanin in biological has been investigated. 5-carboxyfluorescein succinimidyl ester (CFSE) was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic. acid (HI). Various parameters affecting derivatization and CE separation and LIF detection were systematically studied. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical and UV detection, with the detection limits of 10^-8 M and 10^-6M, respectively, the 100-fold and 30 000-fold improvements in sensitivity were achieved by applying LIF detection, respectively. The established method was proven to be the most sensitive4. As a key, important and versatile second messenger molecule, nitric oxide (NO) participates the physiological response of plants to the oxidative stress ignited by some environmental factors such as heavy metals contamination. In the present study, It is measured the real time content of NO from ASME under Cd stress using the present established microplate-based fluorescence detection method. The results obtained showed that the NO increased with the treatment time (0～28 d ) at thetreatment of Cd. The NO amount produced in the leaves of ASME reached the maximum significant difference at 20th day. The NO was proven to be one of the induced products of the physiological response of ASME to Cd stress.5. The synergic enhance effects of the selected three antioxidant tolerance, viz.. melanin, GSH and SA, on the toxicity of Cd on ASME and on its Cd tolerance capacity were investigated by hydroponic experiments. The results showed that, three antioxidant substances enhanced effectively the tolerance abilities of ASME to Cd. The decreasing of the induced free radicals and the antioxidant enzymes in the leaves of ASME were also observed. The contents of melanin in ASME was in the following order: root > leaf > stem, obtained by our proposed CE-LIF method coupled with CFSE derivatization, indicating that melanin was absorbed directly by ASME and participated the antioxidant process of ASME under Cd stress.6. The proper increasing of the nitrogenous, phosphate fertilizer can influence the biomass of plant and the heavy metal absorption ability. The results showed that the proper increasing of nitrogenous and phosphate nutrition could increase the enrichment ability of ASME under complex heavy metals contamination. However, the excessive nitrogenous fertilizer not only inhibited the growth rate, but also decreased the Cd, Zn and Pb contents of ASME. The proper increasing the phosphorus nutrition at the concentration level between 0.025 and 0.25 g kg^-1 could improve the growth rate and the dry weight of ASME, and significantly improved the absorption capacity of ASME on Zn and Pb. However, the continuous increasing of the concentration level of phosphorus fertilizer resulted in the decrease in the absorption capacity of ASME. On the other hand, the application of the KC1 and K₂SO₄ did not improve the biomass and the heavy metal contents.