Uptake, Translocation And Distribution Of Cd And Hg In Brassica Chinensis L.

Environmental pollution of heavy metals,such as cadmium(Cd)and mercury(Hg), from anthropogenic activities are threatening environmental and human health with their unwanted properties.Thus there is an urgent need to develop low-cost,effective, and sustainable methods for their removal and detoxification.Plant-based approaches, such as phytoremediation,are relatively inexpensive and ecologically responsible since they are performed in situ and are solar-driven,while the clean-up methods such as soil washing and electrokinetic remediation removing contaminated soil from a site are generally too costly and environmentally destructive to be applied on the imposing scale that is now required.As metal-hyperaccumulating plants we know mostly have low biomass and poor growth,it is hard to use in phytoremediation. Metal-hyperaccumulating plants with unique abilities to tolerate,accumulate,and detoxify metals represent an important reservoir of unique genes that could be transferred to fast-growing plant species for enhanced phytoremediation.Therefore,it is primary to elucidate the mechanisms by which plants accumulate and detoxify heavy metal.In this thesis,Brassica chinensis L.,which is one of the staple and most popular vegetables in China,was chosen as a model to investigate the biochemical responses of plants under Cd and Hg stress.It consists of 5 chapters as below:In Chapter One,the development of phytoremediation and cellular mechanisms for heavy metal detoxification and tolerance were introduced;the analytical techniques of elemental speciation have also been reviewed.Research proposal was thus made for this thesis,aiming to study Cd and Hg accumulation,speciation and detoxification in B.chinensis.In Chapter Two,B.chinensis was exposed to different concentrations of Cd to evaluate its Cd-accumulating capacity and its potential cellular defensive mechanisms. Cd accumulation in the shoots and roots,respectively,of B.chinensis was up to 1348.3 and 3761.0 mg Kg^-1dry weight under Cd exposure of 200μmol L^-1. Increasing Cd accumulation in the plant was accompanied by rapid accumulation of phytochelatins(PCs),and the sequestration of Cd by PCs seemed to provide a primary cellular mechanism for Cd detoxification and tolerance in B.chinensis.Furthermore, malondialdehyde(MDA)formation,hydrogen peroxide(H₂O₂)content and antioxidative enzyme activities such as superoxide dismutase,catalase,guaiacol peroxidase and ascorbate peroxidase(APX)were observed in the shoots of Cd-stressed B.chinensis.Increasing enzyme activities in response to concentrations from 5 to 50μmol L^-1Cd showed an efficient defense against oxidative stress, suggesting that the antioxidative system was a secondary defensive mechanism.These resulted in reduced free Cd damage and enhanced Cd accumulation and tolerance. However,at higher concentrations of Cd,the depletion of glutathione,the subdued production of PCs and the inhibition of protective enzyme activities,especially APX activity,promoted H₂O₂ accumulation and exceeded the detoxification capacity, leading to cell death.In general,these results suggested that PCs and the antioxidative system are synergistic in combatting Cd-induced oxidative stress and that they play important roles in Cd detoxification.In Chapter Three,in-vitro synthesized Cd-phytochelatin(PC)complexes and in-vivo Cd-PC complexes in Cd-stressed B.chinensis,which has been identified as a Cd hyperaccumulator,were characterized using SEC-ICP-MS and ESI-MS/MS.The PCs(n=1-5)obtained from Cd-stressed B.chinensis together with CdCl₂ were used to synthesize the in-vitro Cd-PC complexes,and the formation of CdGS_1-2,(CdGS)₂, Cd_1-2PC₂,Cd_1-3PC₃,Cd_1-3PC₄ and Cd_1-3PC₅ was observed.In addition,for the first time,in-vivo CdPC₃ and CdPC₄ complexes,as well as Cd-free PCs(n=2-5)and desGlu-PC₃ were detected in the extracts of Cd-stressed B.chinensis and confirmed by means of their corresponding isotopic peak distribution and MS/MS spectra. Nitrogen saturated ammonium bicarbonate buffer(pH=7.8),instead of Tris-HCl and phosphate buffer,was used as a suitable mobile phase in order to stabilize the Cd-PC complexes and effectively avoid possible oxidation of PC analogues during SEC fractionation.Results obtained in this study give definite evidence elucidating the important roles which PCs play in plant defensive mechanisms to Cd stress. SEC-ICP-MS and ESI-MS/MS were demonstrated as powerful and promising techniques for screening and identifying the in-vivo metallopeptides,with accurate isotopic distribution assignment,in metal toxicology studies.In Chapter Four,the accumulation of Hg and the formation of PCs and Hg-PC complexes in B.chinensis plants under HgCl₂,(HgCl₂+humic acids)and HgCys₂ exposure are studied.Treatment with humic acids and Cys has decreased the Hg accumulation in B.chinensis roots but increased seedling growth and induced PCs synthesis in the roots.Speciation studies on in-vitro constituted and in-vivo Hg-PC complexes and PCs were conducted using RPLC coupled on-line with ICP-MS and ESI-MS/MS.The formation of HgGS₂,HgPC₂,HgPC₃,HgPC₄ and Hg₂PC₄ in vitro has been observed and characterized by ESI-MS/MS.Besides oxidized PC_2-4,in-vivo HgPC_2-4and Hg₂PC₄ complexes have also been detected in the roots of humic acids and Cys treated B.chinensis under Hg stress and confirmed by their corresponding accurate isotopic peak distribution and MS/MS spectra for the first time.The preliminary results suggest that humic acid and Cys play an important role in Hg detoxification and tolerance in B.chinensis for not only PCs synthesis but also consequent sequestration of Hg²⁺by forming Hg-thiolate.Intensive studies of the biological mechanisms behind these phenomena should be carried out in the future.In Chapter Five,a summary of this thesis was concluded and the developing trend was also discussed.