Contribution Of Apoplasmic Bypass To Cadmium Uptake And Transport In Hyperaccumulator Sedum Alfredii Hance

Recently,as the Cadmium(Cd)pollution is seriously exacerbated,much attention has been paid to remediation of Cd contamination.Cadmium-hyperaccumulating species,which are capable of accumulating and tolerating up to 100μg Cd g-1 in shoots,are therefore of potential use in the development of phytoremediation approaches for Cd-contaminated soils.Sedum alfredii(Crassulaceae)is one of a few Zn/Cd hyperaccumulators native to China,which has significant potential for use in phytoremediation owing to its high resistance to and hyperaccumulation of Cd.While numerous studies have demonstrated that Cd is symplasmically transported,much less attention has been paid to the contribution of the apoplasmic pathway in Cd hyperaccumulation.Here,we employed a combination of stable isotopic tracer techniques,tissue staining,immunofluorescent staining,high performance liquid chromatography(HPLC),scanning ion-selective electronic technique(SIET),reverse transcriotion-polymerase chain reaction(RT-PCR)and inductively coupled plasma mass spectrometry(ICP-MS)to investigate and quantify the impact of the apoplasmic pathway on translocation of Cd to the xylem in roots of a hyperaccumulating ecotype(HE)and a non-hyperaccumulating ecotype(NHE)of Sedum alfredii as well as the factors affecting apoplasmic pathway.The main results include:1.Cd may reach the xylem either through the symplasmic pathway or apoplasmic bypass(AB);the relative contribution of each pathway in Cd hyperaccumulation in S.alfredii,however,is little understood.The results showed that xylem sap volume and Cd concentration in xylem sap were significantly inhibited by addition of metabolic inhibitor,carbonyl cyanide m-chlorophenylhydrazone(CCCP)in both ecotypes.However the Cd concentration in xylem sap of HE was still as 5 times much as NHE.The rate of 113Cd influx to the shoots exceeded that to the roots in HE,indicating that an AB must contribute to the delivery of Cd to the xylem.This was further proved by the observation that Cd and AB tracer(trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid,PTS)pathway partially overlapped in HE root.Additionally,the Cd content in the xylem sap of HE was proportional to hydrostatic pressure,which was attributed to pressure-driven flow via the AB.The contribution of the AB to Cd transportation to the xylem was increased with increased Cd concentration in HE(up to 37%at the highest concentration used).2.The significance of polysaccharide modification in metal cell wall(CW)of hyperaccumulation is less known.The fractions of CW polysaccharides include pectin,hemicellulose 1(HC1)and hemicellulose 2(HC2),were significantly greater in the NHE than in the HE irrespective of Cd treatment.CW bound Cd was much higher in NHE than in HE,and the pectin was the major site for Cd absorption.The activity of pectin methylesterase were higher in the NHE than in the HE both in the absence or presence of Cd.Additionally,more pectin were methylated in the HE than in the NHE,indicating more free pectic acid residues in the NHE.Cd absorbed was more readily available for transport to the shoots by the HE roots than NHE,which is confirmed by a 6-fold higher 113Cd concentration in xylem sap.The cell-wall-bound Cd was retained more tightly in the NHE than in the HE.Cadmium hyperaccumulation by HE of S.alfredii is associated with its enhanced Cd flux into free space of apoplasmic.which is partly regulated by cell-wall polysaccharide modification in roots.3.Cadmium(Cd)hyperaccumulation in S.alfredii was strictly related to morphogenic response induced by Cd in the root system.Adaptive response included changes of the root apex size and the development of apoplasmic barrier.Cd-treated HE roots showed significantly higher hydraulic conductance than those of NHE,which is in accordance with less extensive suberization due to reduced expression of SaCYP86A1,SaKCS20 and SaCASP.The HE had more extensive meristematic zones and elongation zones than NHE under Cd exposure.The apoplasmic transport,which facilitated Cd and an apoplasmic tracer to reach xylem in HE,was mainly localized in root apexes and lateral roots,where casparian strips and suberin lamellae were not well developed.4.The mechanism of Cd hyperaccumulation with the aid of organic acids secretion has not been well documented.HE had nearly 2-fold higher oxalate secretion than NHE.Phenylglyoxal(PG)effectively blocked Cd-induced oxalate secretion and decreased Cd concentration in HE while exogenous oxalate supply promoted Cd accumulation efficiently.Scanning ion-selective electrode technique(SIET)analysis indicated that Cd2+influx into roots of HE mainly occurred at zone 0-10 mm from root apex where oxalate secretion was localized.Cd was distributed preferentially to the root stele of the HE but not the NHE,and was significantly increased with the secreted oxalates in HE.Exogenous PG effectively induced the development of casparian strips(CS)while addition of oxalate delayed the deposition of CS.In response to the development of CS,the PTS was detected less under well developed CS in NHE,but more under less developed CS in HE.In conclusion,Cd induced oxalic acid from root apex contributed to Cd hyperaccumulation by affecting deposition of CS.5.Abscisic acid(ABA)as one of the most important plant hormone plays a vital role in heavy metal tolerance,while the significance of ABA on Cd uptake and translocation in hyperaccumulators has never been studied.When exposed to Cd,the HE had better water uptake and transport(i.e.further deposited CBs,and less varied endogenous ABA concentration,but higher stomatal conductance,whole plant transpiration and leaf potentials)than NHE,which was correlated with ecotypic difference in plant growth.Higher Lpr and aquaporin protein activity were associated with peak SaPIP expression levels in the root tip of HE than NHE under Cd treatment.An analysis of the endogenous abscisic acid(ABA)variance and exogenous ABA/ABA synthesis inhibitor(Abamine)demonstrated that the endogenous ABA levels was significantly lower in HE than NHE,irrespective of Cd treatment;exogenous ABA treatment clearly decreased Cd accumulation in both shoots and xylem sap of HE,induced closer deposition of suberin lamellae and significant decrease in hydraulic conductance,stomatal conductance,transpiration rates and water status as well,while exogenous supply of Abamine have the opposite effect on NHE,especially under Cd treatment.Taken together,these findings highlight the significance of ABA,as a regulator mediating deposition of apoplasmic barrier and stomatal activity,controlling water balance and Cd hyperaccumulation in hyperaccumulators.