Hyperaccumulator The Southeast Sedum (the Sedum Alfredii Hance In Detoxification Mechanisms) Of Heavy Metals (zn / Cd / Pb)

Heavy metal pollution is a widespread and important environmental concern. Various in situ and ex situ cleanup technologies have been employed, of these methods, phytoremediation is promising due to its low implementation costs and significant environmental benefits. Hyperaccumulator and accumulator species, which efficiently tolerate and accumulate heavy metals from the soil into shoots, have great promise in phytoremediation of contaminated environments. Understanding the mechanisms of metal tolerance and accumulation will provide insight into the identification and management of these hyperaccumulating species. Sedum alfredii Hance (Crassulaceae) is the recently identified Zn/Cd co-hyperaccumulator native to Pb/Zn rich regions of China. This species exhibits a strong ability to both tolerate and accumulate considerable amounts of Pb. A better understanding of the biological mechanism involved in heavy metal accumulation in this species would help to improve its practical application. The aim of this study was to obtain fundamental information on heavy metals accumulation and tolerance mechanisms in S. alfredii, by investigation of the metal localization and ligand abundance using several techniques. The main results include:1. Short-term responses of roots of the Cd hyperaccumulating ecotype (HE) S. alfredii to Cd exposure were compared with its non-hyperaccumulating ecotype (NHE). Marked root elongation inhibition was observed in roots of NHE in response to Cd exposure, while such inhibition was much slighter in HE, and stimulation of root elongation was observed with 10μM Cd treatment. The loss of plasma membrane integrity and lipid peroxidation in roots tips of NHE increased significantly with Cd treatments, whereas these effects of Cd was only pronounced at 400μM in HE root tips. A strong dose-dependent accumulation of reactive oxygen species (ROS) with increasing Cd was noted in the NHE root tips, but not in roots of the HE plants. In response to Cd exposure, GSH increased in the HE root tips but tended to decrease in those of NHE. A dose-dependent decrease in GSSG and an increase in NPT caused by Cd were marked in root tips of HE, but were not seen in the NHE plants. These results suggested that in contrast to the NHE, the HE S. alfredii tolerates high Cd in the environment and maintains root function through the differential expression in a number of hypertolerance mechanisms including altered glutathione metabolism, reduced ROS accumulation and hence prevent lipid peroxidation, loss of plasma integrity, and inhibition of root elongation.2. The characteristics of in vivo Zn distribution in stems and leaves of the HE and NHE S. alfredii were investigated by synchrotron radiation X-ray fluorescence analysis, together with a Zn probe. Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaf and stem of HE as exposure time prolonged, while Zn saturation occurred relatively early in its leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. Leaf and stem distribution patterns of P and S in the HE were very like that for Zn, while the Ca distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. Our study mainly, suggested that epidermal layers serve as large storage sites for the excess Zn in the hyperaccumulator HE S. alfredii:3. The spatial information of Cd distribution within the leaf of HE S. alfredii was obtained by LA-ICP-MS together with XRF. The results showed that Cd was preferentially distributed in the veins of the whole leaf, whereas the content of the element in the leaf edge is relatively low. The localization of Zn appears to be the reverse of that for Cd. By analysis of the leaf cross-section using XRF, it is found that Cd was largely localized in the vascular bundles and upper epidermis, whereas its content in the lower epidermis is lower. Zinc was extremely concentrated in the epidermis, indicating a very different sequestration of the two elements within the leaf of HE S. alfredii.4. To investigate the spatial distribution of Pb in the accumulator plant, micro scanning XRF mapping was performed on both Accumulating ecotype (AE) and Non-accumulating ecotype (NAE) S. alfredii. The leaf cross-section image of AE revealed that Pb was accumulated mainly in the leaf veins, with a second peak in the epidermis. Much lower content of Pb was found in either spongy or palisade mesophyll cells. An almost exclusive localization and accumulation of Pb within vascular bundles was noted in the AE stem. Distribution patterns of S in both leaf and stsem cross-sections were quite similar to that of Pb, and a significant positive correlation between the XRF intensities of Pb and S was observed, whereas no such correlation were observed NAE plants. Besides, addition of EDTA in the culture solution did not change the preferential distribution of Pb in the vascular bundles, although the intensity of Pb was much lower, as compared with those with Pb alone.5. Zinc speciation in the different tissue and xylem sap of HE and NHE S. alfredii were determined by EXAFS and HPLC. The results showed that the majority of Zn in all tissues was coordinated with oxygen or nitrogen ligands both two S. alfredii ecotypes. After 24 h exposure to 100μM Zn, Zn speciation in the xylem sap of HE occurred in three forms, including Zn2+, Zn-Ctrate and Zn-Malate, of 55.87%,42.31% and 6.07%,respectively. Analysis of organic acids in the xylem sap of the plants with Zn treatments supported the possible role of citrate in Zn speciation. Abundant Citrate occurred constitutively, and increased by Zn exposure in the xylem sap of both ecotypes of S. alfredii. These results suggested that Zn-Citrate complex might play an important role in Zn hyperaccumulation of HE S. alfredii.6. Cadmium speciation and organic acid concentration in the different tissue of HE S. alfredii were determined by using EXAFS and HPLC, respectively. The results showed that the majority of organic acids in the leaf was malate, and malate concentration in the shoot was affected by increasing Cd concentration in the solution, suggesting that malate synthesis is induced by Cd. In root, Cd was coordinated by O/N and S ligands in the first shell, which indicates strong ligands are required for metal detoxification. In young leaf and mature leaf, Cd was coordinated by O/N and O in the fisrt shell and by carbon ligands in the seconds shell.7. In vivo speciation of Pb in tissues of the accumulator plant, was investigated using powder-and micro-X-ray absorption near edge structure (XANES) spectroscopy. The dominant chemical form of Pb in tissues of both accumulating (AE) and non-accumulating ecotype (NAE) S. alfredii was similar to previously defined Pb-cell wall compounds, though a much higher percentage of the Pb in the stem and leaf of AE appeared to be associated with SH-groups (GSH). Micro-XANES analysis of tissue cross sections further verified that Pb in the epidermis and pith were indicative of Pb-cell wall and Pb-pectin complexes, whereas Pb within vascular bundles was predominantly Pb-GSH complex, suggesting that this species may transport Pb via a Pb-GSH complex. By application of EDTA, the complexation of Pb-EDTA was detected in the plant samples, indicating the speciation of Pb within the plants may also change due to the environment circumstance.