Speciation Analysis And Distribution Of Cadmium And Their Role In Cadmium Detoxification Of Indian Mustard By Hyphenated Mass Spectrometric Techniques

Cadmium (Cd) pollution in the soil is one of the most significant global environmental problems, as it will enter into human bodies through the accumulation of the food chain, and endangers the health of human being. 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. Because of the analytical limit, there has not yet a common accepted conclusion on the migration mechanism of the heavy metal within the plant, as well as the detoxification mechanisms. Phytochelatins (PCn), characterized by the amino acid structure (y-Glu-Cys)n-Gly, where n ranges from2to11, are synthesized enzymatically by phytochelatin synthase (PCn) using glutathione as a substrate in the presence of many metals and metalloids.They are believed to play an important role in metal detoxification and tolerance. But few research on speciation analysis of PCn-Cd and its unstability on detoxification mechanism has been studied.Therefore, in order to reveal migration and transformation pattern of heavy metals and investigate the relationship between speciation and tolerance of heavy metals, some studies on Indian Mustard which was demonstrated to tolerate and accumulate considerable amounts of cadmium were carried out. The aim was to establish methods for speciation and distribution analysis of cadmium, and obtain fundamental information on heavy metals accumulation and tolerance mechanisms by investigation of the metal localization and ligand abundance. Several modern hyphenated mass spectrometric techniques, such as high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), ultra performance liquid chromatography-electrospray ilonization mass spectrometry (UPLC-ESI-MS), ultra performance liquid chromatography-time of flight mass spectrometry (UPLC-QTOFMS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were used.The main results include:1. Exposure concentration of Cd above1.0mg/L will bring negative effects on the normal growth of the plant, and therefore result in the decrease of the biomass. Within48hours after the stimulation, the symptoms of intoxication can be observed in the plant. With the increase of the exposure time and concentration, the concentrations of cadmium in root, leaf and stem also increases. The concentration of cadmium in the root is far more than that in stem and leave, and and the lowest in in leave. 2. In this study, in vitro formed PCn-Cd complexes were characterized using UPLC-ESI-MSMS and UPLC-QTOFMS. These two methods have been demonstrated to be ideal and promising techniques for screening and characterizing peptide-metal complexes. It was demonstrated that both techniques had the ability to identify the formation of PCn-Cd complex.Furthermore, the increase of PC after Cd exposure demonstrated that Cd is the most efficient inducer of phytochelatin synthesis.3. Method based on the coupling of size-exclusion HPLC with ICP-MS was used for speciation analysis of cadmium binding non-protein thiols in extracts of Indian mustard (Brassica Juncea). In the presence of Cd stress, PCn were induced, and Cd ions in extracts were associated with the induced PCn. So four different Cd species were detected in samples:PC3-Cd、PC2-Cd、GSH-Cd and Cys-Cd, which demonstrated that PCn were enzymatically synthesised from glutathione (GSH, Glu-Cys-Gly) by the constitutive enzyme PC synthase. The results indicated that GSH-Cd exists as the main specie in leaf samples while PC2-Cd is the main specie in root samples. A non-oxidizing (Nitrogen) preparation environment and a-70℃preservation condition were employed to avoid the oxidation of sulfhydryl groups.4. Root and leaf samples were submitted to0(control),2.0,4.0,6.0,8.0,10.0mg L-1and harvested after1,2,3and4days exposure. Results indicated lower Cd stress levels were generally associated with higer PCn-Cd production. Higher stress induced the reduction of PCn-Cd, which proved that thiol based tolerance was only efficient in lower degrees of stress. Increasing exposure time also induced decreased PCn-Cd pordution indicated PCn may only had a partial role in metal resistance. Under continuous higher stress, plants maybe trigger other mechnisms to tolerate heavy metal toxicity. Our results suggested the concentration and time of exposure are important factors that must be taken into consideration when evaluating the true role of PCn in heavy metal detoxification.5. Method based on the coupling of size-exclusion HPLC with ICP-MS was used for speciation analysis of Cd, Cu and Zn binding non-protein thiols in extracts of Indian mustard(Brassica Juncea). In the presence of Cd, Cu and Zn stress, PCn were induced, and metal ions in extracts were associated with the induced PCn. So four different species were detected in stem and root:PC3-Cd(Cu,Zn), PC2-Cd(Cu,Zn), GSH-Cd(Cu,Zn) and Cys-Cd(Cu,Zn). Moreover, results indicated the existence of complexation competition for GSH and cysteine between Cd, Cu and Zn.6. Imaging of trace metal distribution in tissue sections by laser ablationinductively coupled plasma-mass spectrometry (LA-ICP-MS) is typically performed using spatial resolutions of25μm, including Cd, P,S, K,Ga,Gu-and Zn elements. The-results showed that Cd was preferentially distributed in vascular tissue. The high relationship between Ca and Cd distribution indicating a very similar sequestration of the two elements within Indian Mustard.7. Method based on ICP-MS was used for subcellular distribution of cadmium in extracts of Indian mustard(Brassica Juncea). Brassica Juncea were submitted to0,0.5,1.0,3.0,5.0,10.0mg L-1and harvested after1,5,7,10and14days exposure. Cells were separated into three fractions:cell wall, soluble fraction and organelle containing fraction using differential centrifugation technique. Results indicated that higher subcellular cadmium in both leaf and root samples was found as exposure level and exposure time increase, but Cadmium concentration differs slightly under1.0mg/L exposure. Results of TEM under different exposure also demonstrate it. Subcellular fractionation of Cd-containing tissues indicated that about50-62%of the element was localized in cell walls and22-32%in soluble fraction, and the lowest in cellular organelles. It could be suggested that subcellular compartment played an important role in Cd detoxification.