| An economic boom brings us a worsening problem of heavy metal pollution. As a new environment-friendly and low-cost technique, utilizing plants to transport, contain and transform the pollutants from the environment, in-situ technique shows a widely applied prospect in the ecological remediation. Hyperaccumulator is a specific plant with its aboveground parts rich in heavy metal. So it is a good material for ecological restoration. A further study of the Hyperaccumulators can not only promote the development of plant nutrition theory but also help for lay the foundation of phytoremediation of heavy metal contaminated soil technology. However, at present, the research is focused more on crucifer, especially on hyperaccumulators of Ni/Zn compared to those of Cd. Sedum alfredii Hance is an original Chinese plant hyperaccumulated of zinc, Cd and Pb, has a potential application prospect in the field of soil remediation in China. Although there are some studies on hyperaccumulating characteristics and physiological mechanism of Sedum alfredii Hance, the mechanism of its absorption and accumulation of Cd are still not well-understood. Previous research shows that the rapid uptake by the roots, loading by xylem and the segmentation effect of shoot play the major role in the process of Cd accumulation for Sedum alfredii Hance. Furthermore the absorption, accumulation and detoxification mechanisms of Cd have a close relation with the absorption and metabolic pathway of Ca. However, we still do not know how Sedum alfredii accumulate and detoxify Cd in its aboveground parts. And the relationship between it and the Ca transport system also beyond our knowledge. So, based on establishing and improving the technology of the protoplast isolation and purification for leaves, by using HE and NHE Sedum alfredii and Micro-XRF, fluorescence probe positioning and other methods, we study on the relationship between the Ca transport system and the mechanism of absorption and accumulation of Cd by cellulars of aboveground parts. The main conclusions and results of this research are as follows:1. By using Sedum alfredii in nutrient solution culture as raw material, we established and improved the technology of the protoplast isolation and purification for leaves. The results shows that the preheating treatment of the cell lysate can accelerate cell wall degradation, shorten the time of the protoplasts release, and the 2h is the best pyrolysis time for both HE and NHE. We can get more and better protoplast after the treatment of these two methods. On this basis, by comparing parameters, we improve the protoplast lysate, cell protection solution concentration and the steps of the gradient centrifugation. We established the extraction technology of vacuole for these two ecotypes of Sedum alfredii. The technology we established and improved has the advantage as short extracting time, high efficiency, simple operation and etc. Furthermore the protoplasts and vacuoles we obtained have the characteristics of high purity, long survival time etc. The establishing of this method can provide technical support for the further study of the Sedum alfredii’s heavy metal uptake and accumulation mechanism at cellular and sub cellular levels.2. By using Sedum alfredii in nutrient solution culture for 2 weeks as raw material, obtained the protoplast suspension by using the method we mentioned before, use ICP-MS, fluorescence probe positioning and other methods, we study and compare the characteristics of Cd absorption in two ecotypes of Sedum alfredii leaf cells. We also studied the effect of low temperature, metabolic inhibitors and Ca channel inhibitor. The results show that the Characteristics of the time kinetics and absorption kinetics for Cd absorption has no significant differences between the HE and NHE Sedum alfredii. And the uptake is both inhibited by low temperature. But there is a significant difference between the HE and NHE Sedum alfredii as we found that after the treatment for 10 min-2.0 h by 200 μM Cd, the Cd can be obviously found in the vacuole, but there is no Cd can be found in NHE Sedum alfredii’s vacuole. In addition, after the treatment for 5 h by 200μM Cd, there are still some protoplasts of HE Sedum alfredii remain intact while almost no single protoplasts remain intact for NHE Sedum alfredii. And after Cd treatment at different time (0.25-7.5 h), the survival rate of NHE Sedum alfredii is significantly lower than the HE Sedum alfredii(P<0.05). Moreover, Ca channel inhibitors show the inhibitory effect for HE Sedum alfredii accumulate Cd, while it has no significant effect for NHE Sedum alfredii, it may shows that the absorption of Cd may be regulated by Ca channel. The results showed that there is no significant differences between HE and NHE Sedum alfredii for absorb Cd, and they are both active energy requiring process. The may difference is that the NHE Sedum alfredii’s leaf can compartment the Cd to the vacuole faster, which may slow down the Cd toxicity to cells in a certain extent.3. Based on the preliminary suppose that the absorption of Cd may be regulated by Ca channel for NHE Sedum alfredii and using Micro-XRF technique, we study the influence of different Ca concentration on the distribution and accumulation characteristics of the HE Sedum alfredii at cell level. The result has revealed that Cd accumulation in HE Sedum alfredii leaf is related to Ca treatment and leaf age and the content of Cd and Ca increase with the age of leaf while that in stem is not related to stem age but to Ca treatment (0.5 mM,2.0 mM and 8.0 mM). In addition, as exogenous Ca ion concentration increases, the accumulation of Cd in leaf and stem decreases. Organization maturity has significant treatment effect on Cd accumulation in stem but no significant effect in leaf. Furthermore the distribution image of Cd shows that the low Ca treatment in leaves is significantly higher than that in normal Cd or abundance Ca treatment, and for young leaves the mesophyll cells are the main place for storage Cd. Distribution characteristics of Cd in stem sections shows that the abundance of Cd in vascular tissue at low Ca treatment conditions is several times higher than the normal or high Ca treatment. Furthermore the fine scanning of pipe stem tissue showed that under the condition of low Ca treatment, phloem has the highest abundance of Cd, while at normal Ca conditions, xylem is the place where has the most Ca. The results show that, the low Ca stress induces the transportation of Cd at Sedum alfredii’s phloem and also promotes the absorption and accumulation of Cd in the tender tissues. These results confirm that there is a close relation between absorption, transportation, accumulation of Cd in HE Sedum alfredii and the Ca transport system from issue and cell level. |
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