Cadmium Uptake And Translocation In The Hyperaccumulator Sedum Alfredii H

Hyperaccumulation of Cd by higher plants,defined as being capable of accumulating and tolerating up to 100mg Cd kg^-1in shoots,is a very rare phenomenon.To optimize the potential use of Cd hyperaccumulators for phytoremediation,basic information on the physiological,biochemical and molecular mechanisms of Cd hyperaccumulation is necessary.Information on the rate of uptake and transport of Cd within the plants is essential to better understand the physiology of Cd accumulation in hyperaccumulators.Sedum alfredii is a naturally selected Zn/Cd hyperaccumulator belonging to the Crassulaceae family, which has significant potential for use in phytoremediation.Previous research has revealed that the hyperaccumulating ecotype(HE)of S.alfredii collected from an old Zn/Pb mining area accumulated Cd concentration of up to 9000 and 6500μg g^-1in leaves and stems,respectively,while its contrasting population from an uncontaminated site,non-hyperaccumulating ecotype(NHE),showed severe phytotoxicity at 100μM Cd exposure.Currently our understanding of the mechanisms of Cd hyperaccmulation by plants has focused exclusively on the well-known Cd hyperaccmulators,T. caerulescens and A.halleri,which are both Brassicaceae.A better understanding of the characteristics and physiological mechanisms by which S.alfredii(Crassulaceae)accumulates Cd,may provide additional basic information to aid in the development of these species for phytoremediation purposes. In this study,physiological mechanisms of Cd uptake and translocation in the hyperaccumulator HES. alfredii,in comparison with NHE,were investigated by a combination of several techniques including EXAFS,HPLC,radioactive techniques and ICP-MS,under hydroponic culture.1.By several hydroponic experiments,characteristics of Cd uptake and translocation were investigated in the hyperaccumulating ecotype(HE)of S.alfredii along with its representative non-hyperaccumulating ecotype(NHE).The results showed that measured Cd uptake in HE was constantly much higher than that calculated from transpiration rate.Low temperature(4℃)treatment significantly inhibited Cd uptake in HE plants,whereas no such effect was observed in NHE.In HE, large amounts of Cd was translocated to the shoots,while the translocation rate was markedly suppressed by low temperature.Cadmium concentration was much higher in xylem sap of HE,as compared with that in external uptake solution,whereas opposite results were obtained for NHE.Cd concentration in xylem sap of HE was significantly reduced by addition of metabolic inhibitors, carbonyl cyanide m-chlorophenylhydrazone(CCCP)and 2,4-dinitrophenol(DNP),in the uptake solutions.Furthermore,inhibition of transpiration rate in the HE has no essential effect on Cd accumulation in shoots of the plants.These results suggest that Cd uptake and translocation is an active process in plants of HE S.alfredii,symplastic pathway rather than apoplastic bypass contributes greatly to root uptake,xylem loading and translocation of Cd to the shoots of HE,in comparison with the NHE plants. 2.A combination of radioactive techniques,metabolic inhibitors and fluorescence imaging was used to contrast Cd uptake and translocation between hyperaccumulating(HE)and non-hyperaccumulating ecotypes(NHE)of S.alfredii.The K_m of¹⁰⁹gcd-influx into roots was similar in both ecotypes while the V_maxwas 2-fold higher in the HE.Significant inhibition of Cd uptake by low temperature or metabolic inhibitors was observed in HE,whereas the effect was less pronounced in NHE.¹⁰⁹Cd influx into roots was also significantly decreased by high Ca in both ecotypes.The rate of root-to-shoot translocation of ¹⁰⁹Cd in HE was over 10 times higher when compared with NHE and shoots of HE accumulated dramatically higher ¹⁰⁹Cd concentrations than shoots of NHE.The addition of the metabolic inhibitor CCCP resulted in a significant reduction in Cd contents in the shoots of HE,and in the roots of NHE Cadmium was distributed preferentially to the root cylinder of HE but not NHE,and there was a 3-5 times higher Cd concentration in xylem sap of HE in contrast to NHE.These results illustrate that a greatly enhanced rate of root-to-shoot translocation,possibly as a result of enhanced xylem loading, rather than differences in the rate of root uptake,was the pivotal process expressed in the Cd hyperaccumulator HES.alfredii.3.Cadmium speciation in the xylem sap of HE and NHE S.alfredii was determined by EXAFS,the results showed that the majority of the Cd in the xylem sap was coordinated with oxygen or nitrogen ligands both two S.alfredii ecotypes,however,some of Cd-S complex was observed exclusively in the xylem sap of HE.After 24h exposure to 100μM Cd,Cd speciation in the xylem sap of HE occurred in three forms,including Cd²⁺,Cd-oxalate and Cd-GSH,of 62.57%,28.21%and 11.18%,respectively.As exposure time prolonged to 7d,percentage of Cd²⁺increased,with the other two forms decreased in the xylem sap of HE.Free ionic forms of Cd and Cd-oxalate were also existed in the xylem sap of the contrasting ecotype(NHE),while a few of Cd was found to be associated with citric acid.Analysis of organic acids in the xylem sap of the plants under Cd stress supported the possible role of oxalate in Cd speciation of S.alfredii.Abundant oxalic acid occurred constitutively,and increased by Cd exposure in the xylem sap of both ecotypes of S.alfredii.These results suggested that Cd occurred mainly in the free ionic form in the xylem transport of both the two ecotypes,with some coordinated with oxalate, whereas Cd-S complex might play an important role in Cd hyperaccumulation of HES.alfredii.4.Effects of divalent ions,in particular of Ca and Zn,on root uptake and translocation of Cd in HE and NHE S.alfredii were investigated,as to reveal the possible pathway of Cd entry into the plants. The results showed that a statistically significant inhibition of ¹⁰⁹Cd influx into the roots of both two ecotypes was induced by high Ca treatment,whereas no such effect of other divalent ions were observed.Under high Ca exposure,the V_maxof ¹⁰⁹Cd-influx into roots was 2-fold higher in the HE while the V_maxremained at similar level,when compared with those of the control.Contrast results were observed in the NHE,with its similar Km but dramatically decreased V_maxinduced by Ca.Long time exposure to high Ca was found to reduce the Cd toxicity in both two ecotypes.Both total cadmium influx and root-to-shoot translocation in the NHE was reduced by high Ca,whereas translocation rate of Cd increased significantly with the increasing Ca treatments.Zinc and Ca concentrations in the xylem sap of the both ecotypes decreased with the increasing external Cd level, but significant negative correlationships between Cd and Zn/Ca was observed only in the xylem sap of the HE.Increased xylem transport of Cd was observed in the HE by addition of appropriate exogenous Zn/Ca.These results suggest root uptake and translocation of Cd in the NHE plants is mainly through Ca transporters or channels,whereas a multi-transport system exists within the plants of HE,but Ca/Zn pathway was,at least partly,involved in the Cd accumulation of the hyperaccumulator.5.The impact of exogenous tartaric acid and citric acid on Cd uptake,root-to-shoot translocation and accumulation of the HE Sedum alfredii were investigated.The results showed that short-term(2h)root influx of ¹⁰⁹Cd increased significantly by addition of tartaric acid or citric acid.After 24h exposure, ¹⁰⁹Cd in both root and shoot of the plants was 1.5 and 3.0-fold higher under citric acid treatment,when compared with the control,and a significant increased root-to-shoot translocation of ¹⁰⁹Cd induced by citric acid was also observed(P＜0.05),whereas the main effect of tartaric acid was increased ¹⁰⁹Cd influx into the roots of the plants.Cd toxicity of the plants was reduced by long-term exposure to citric acid,whereas opposite effect of tartaric acid was observed.Long-term exposure to appropriate citric acid or tartaric acid is able to increase the Cd accumulation in the plants of HE,but the effect varies with different exposure time and treatment levels.These results indicates that citric acid may be involved in the Cd uptake and translocation in the HES.alfredii,and increases the tolerance ability of Cd in the plants,whereas the impact of tartaric acid is mainly on the root uptake of Cd Thus, application of the both organic acids may facilitate the phytoextraction of Cd-polluted sites by the hyperaccumulator S.alfredii.