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1 Chemosphere 84 (211) Contents lists ville t SieneDiret Chemosphere journl homepge: Clium protets roots of Sedum lfredii H. ginst dmium-indued oxidtive stress Shengke Tin,, Lingli Lu,, Jie Zhng, Ki Wng, Ptrik Brown, Zhenli He, Jun Ling, Xioe Yng, MOE Key Lortory of Environment Remedition nd Eosystem Helth, College of Environmentl nd Resoures Siene, Zhejing University, Hujihi Cmpus, Hngzhou 3129, Chin University of Florid, Institute of Food nd Agriulturl Siene, Indin River Reserh nd Edution Center, Fort Piere, FL 34945, USA Deprtment of Plnt Sienes, University of Cliforni, Dvis, CA 95616, USA rtile info strt Artile history: Reeived 4 Novemer 21 Reeived in revised form 12 Ferury 211 Aepted 23 Ferury 211 Aville online 21 Mrh 211 Keywords: Clium Cdmium Hyperumultor Oxidtive stress Root Sedum lfredii Sedum lfredii is well-known Cd (dmium) hyperumultor ntive to Chin. The impts of exogenous C on Cd-indued oxidtive stress nd ntioxidnt systems in roots of S. lfredii were investigted y using ellulr nd iohemil pprohes. Supplementtion of the medium with higher C levels resulted in llevited growth inhiition nd deresed Cd onentrtion, s well s inresed C onentrtion in roots. Cdmium indued lipid peroxidtion nd loss of plsm memrne integrity, retive oxygen speies overprodution, s well s ultrstruturl hnges of root ells were lrgely reversed y C supplementtion in the medium. Clium pplition signifintly ltered the Cd effets on ntioxidnt enzymes nd non-enzyme ntioxidnts (non-protein thiols), nd signifintly inresed glutthione (GSH) iosynthesis. The results suggest tht C is le to protet the roots of S. lfredii ginst Cd toxiity y restortion of Cd-displed C, llevition of the metl indued oxidtive stress, s well s promotion of GSH iosynthesis. Ó 211 Elsevier Ltd. All rights reserved. 1. Introdution Industril proesses nd widespred use of phosphte fertilizers hve resulted in n inrese in dmium (Cd) in the environment over the pst dedes (Pinto et l., 24). Due to its high moility nd toxiity inresed environmentl Cd represents thret to plnt nd niml helth (Bertin nd Averek, 26). Cdmium is potent toxi element for plnts, using root tip dmge, redued photosynthesis, indued ntioxidnt responses in ll plnt orgns, nd growth inhiition (Ds et l., 1997). A rre lss of plnts nmed hyperumultor (Bker et l., 2), however, n tolerte nd umulte n extremely high onentrtion of Cd in the oveground tissues nd hve potentil for use in phytoremedition of metl-polluted soils (Pilon-Smits, 25), nd onstitute n exeptionl iologil mteril for understnding mehnisms regulting plnt metl homeostsis s well s plnt dpttion to Arevitions: CAT, tlse; DHE, dihydroethidium; DTNB, 5,5 -dithiois-2- nitroenzoi id; GSH, glutthione; G-POD, gluthione peroxidse; MDA, mlondildehyde; NPT, non-protein thiols; ROS, retive oxygen speies; SOD, superoxide dismutse; TBARS, thiorituri id retive sustnes; TCA, trihloroeti id; TMP, tetrmethyl piperidinooxy. Corresponding uthors. Tel.: ; fx: (L. Lu), tel./fx: (X. Yng). E-mil ddresses: linglilulu@gmil.om (L. Lu), xyng571@yhoo.om (X. Yng). extreme metlli environments (Verruggen et l., 29). Sedum lfredii is fst growing, high-iomss Zn/Cd ohyperumultor nd P umultor ntive to Chin (Lu et l., 28; Tin et l., 29, 21). The mehnisms involved in the metl umultion y this plnt speies, however, re not well understood. Metl hyperumultion in plnts is generlly hieved y omintion of enhned metl uptke nd trnslotion, oupled with etter tissue tolerne to the phytotoxiity of the elevted metls (Jin et l., 28). Cdmium is non-essentil element for plnts nd its uptke is thought to our primrily through trnsporters for essentil elements s onsequene of lk of speifiity of the trnsporters (Welh nd Norvell, 1999). Clium shres mny physil similrities with Cd nd hs similr hrge nd ioni rdius t physiologil ph vlues, nd ntgonism etween the two elements hs een frequently reported in plnts. Cdmium ompetes with C for uptke through ion hnnels in root ells nd gurd ells of plnts (Clemens et l., 1998; White, 2; Perfus-Breoh et l., 22). In previous studies, we found tht ptterns of Cd nd C uptke, trnslotion nd distriution in the hyperumultor S. lfredii were similr (Lu et l., 28, 21) nd supplementtion of C in the growth medium resulted in elevted Cd umultion in shoots nd improved plnt growth of S. lfredii (Lu et l., 21). The mehnisms involved in the C llevition of Cd toxiity, however, re lrgely unknown /$ - see front mtter Ó 211 Elsevier Ltd. All rights reserved. doi:1.116/j.hemosphere

2 64 S. Tin et l. / Chemosphere 84 (211) The present investigtion ttempts to identify the mehnisms y whih C protets S. lfredii from Cd stress. It is well known tht Cd uses disturnes to plnt ntioxidnt defenses, nd indiretly triggers the prodution of vrious retive oxygen speies (ROS) y unknown mehnisms, giving rise to n oxidtive urst in plnts (Olmos et l., 23; Romero-Puerts et l., 24; Grnier et l., 26; Rodriguez-Serrno et l., 29). Clium ws shown to e involved in the signl trnsdution of environmentl stimuli nd relted gene expression in plnts, inresing the tolerne of plnts ginst Cd nd other stresses (Wng nd Song, 29). The protetive effets of C ginst Cd-indued oxidtive stress hve now een reported in severl plnt speies (Rodriguez-Serrno et l., 29; Wng nd Song, 29). The present investigtion ws performed to determine if supplementl C protets the roots of S. lfredii ginst Cd-indued oxidtive stress nd to determine if this ours through effets on ROS tivity (H 2 O 2 nd O 2 ), glutthione metolism, lipid peroxidtion nd the susequent loss of plsm integrity. The study will fous on root tips omprising meristem, elongtion nd differenting ells within short distne of the root tip. Root tips re very metolilly tive nd extremely sensitive to ioti or ioti stress. 2. Mterils nd methods 2.1. Plnt ulture The hyperumultor S. lfredii ws otined from n old P/ Zn mine re in Zhejing Provine, Chin. Plnts were grown in non-ontminted soil for severl genertions to minimize the internl metl ontents, uniform nd helthy shoots were then seleted, rooted nd ultivted in the sl nutrient solution ontining: 2 mm C 2+, 4 mm NO þ 3, 1.6 mm K+,.1 mm H 2 PO 4,.5 mm Mg 2+, 1.2 mm SO 2 4,.1 mm Cl, 1lM H 3 BO 3,.5 lm MnSO 4, 1lM ZnSO 4,.2 lm CuSO 4,.1 lm (NH 4 ) 6 Mo 7 O 24, 1 lm Fe EDTA. Nutrient solution ph ws djusted dily to 5.8 with.1 N NOH or.1 N HCl. Plnts were grown under glsshouse onditions with nturl light, dy/night temperture of 26/2 C nd dy/night humidity of 7/85%. The nutrient solution ws erted ontinuously nd renewed every 3 d. After growing for 14 d, plnts of S. lfredii were sujeted to tretments inluding (1) ontrol, (2) 6. mm C [C(NO 3 ) 2 ], (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd, in nutrition solution (ph 5.8) for 24 h. Three replite were used nd nlyzed independently for eh tretment Root elongtion inhiition Root length mesurements were performed ording to Shutzenduel et l. (21). Roots were mrked 5 mm ehind the tips with wter-resistnt ink 2 d efore tretments. Root lengths were mesured dily on 2 plnts per tretment until hrvest. The degree of the root elongtion (perentge of ontrol) ws estimted s: (B/A) 1, where A ws the root length elongted during tretment without Cd nd B ws the root length elongted during tretments with Cd Determintion of dmium ontent At hrvest, intt roots were soked in 2 mm N 2 -EDTA for 15 min to desor Cd 2+ from the root surfes. After rinsing nd lotting dry, root tips were ut 1 mm ehind the pex, weighed, nd digested with HNO 3 HClO 4. The digest ws mde to volume nd filtered. Conentrtions of Cd nd C in the filtrtes were nlysed using Indutively Coupled Plsm Mss Spetrosopy (ICP-MS) (Agilent 75, USA) Loss of plsm memrne integrity nd lipid peroxidtion Plsm memrne integrity ws mesured spetrophotometrilly with Evns lue ording to Ymmoto et l. (21). After tretment pplition, thirty root tips (1 mm) were inuted in Evns lue solution (.25% [w/v] in 1 lm CCl 2 (ph 5.6) solution) for 3 min. After wshing the roots for 15 min with wter, roots tips were homogened with mirohomogenizer in 2 ml of mesuring solution (5% [v/v] MeOH nd 1% [w/v] SDS). The homogente ws inuted for 15 min in wter th t 5 C nd entrifuged t 14 g for 15 min. The optil density of the superntnt ws determined t 6 nm. The level of lipid peroxidtion in root tips ws determined s thiorituri id retive sustnes (TBARS), nd mlondildehyde (MDA), sed on the method of Dixit et l. (21). Briefly, pproximtely thirty root tips (weighed) were ground with 5 ml of.1% trihloroeti id (TCA). The homogente ws entrifuged t 15 g for 1 min nd.5 ml of the superntnt ws mixed with 2 ml of.5% TBA in 2% TCA. The mixture ws heted t 9 C for 2 min. After the retion ws stopped, the resultnt mixture ws entrifuged t 1 g for 5 min. The sorne of the superntnt ws mesured t 532 nm. The vlues were orreted for non-speifi sorption y sutrting sorne red t 6 nm. The mount of TBARS ws lulted y using the extintion oeffiient of 155 mm 1 m 1. Histohemil detetion of loss of plsm memrne integrity nd lipid peroxidtion in root pies ws performed s desried y Ymmoto et l. (21). Intt roots of S. lfredii seedlings fter different tretments were rinsed severl times with.5 mm CCl 2 (ph 4.5), dried with filter ppers. For plsm memrne integrity determintion, roots were inuted in 4 ml of Evns lue solutions (.25% (w/v) prepred in 1 lm CCl 2 (ph 5.6) for 3 min. For lipid peroxidtion nlysis, roots were inuted in Shiff s regent for 6 min, rinsed with solution ontining.5% (w/v) K 2 S 2 O 5 (prepred in.5 M HCl) until the root olor eme light red. All of the roots, stined with the speifi regents s indited ove, were wshed three times with wter nd oserved under light mirosope (model SZH-ILLD; Nikon, Jpn) ROS (H 2 O 2 nd O 2 ) ontent ssy nd fluoresene imging The ontent of H 2 O 2 ws mesured ording to the method of Jin et l. (28) with smll modifitions. Root tips were ground in 5 mm K-phosphte uffer (ph 7.8). To the homogente, 5% TCA ws dded (TCA: mixture/1:.7). The mixture ws entrifuged t 1 g for 1 min. nd the superntnt ws olleted. One ml of the superntnt ws dded to 1 ml of 1 mm potssium phosphte uffer (ph 7.) nd 2 ml of 1 M KI. H 2 O 2 onentrtion ws estimted sed on the sorne of the superntnt t 39 nm. Determintion of O 2 prodution in root tips of plnts were mesured s desried y Hung et l. (28). Root tips smples were ground with ie-old sodium phosphte uffer (ph 7.8, 5 mm). The extrts were entrifuged t 13 g for 2 min t 4 C. The superntnt (.5 ml) ws olleted nd inuted t 25 C for 6 min in the presene of 1 mm hydroxylmine hydrohloride in 5 mm sodium phosphte uffer (ph 7.8). The retion mixture ws then inuted with 1 ml of 17 mm P-minoenzene sulphoni id nhydrous (dissolved with eti id: H 2 O 2 = 3:1) nd 1 ml of 7 mm -nphthylmine (dissolved with eti id: H 2 O 2 = 3:1) t 25 C for 3 min. The sorne ws mesured t 53 nm. A lirtion urve ws estlished using sodium nitrite. Intt roots of S. lfredii seedlings fter different tretments were rinsed severl times with.5 mm CCl 2 (ph 4.5), dried with filter ppers, nd immeditely immersed into the following speifi regents. Superoxide rdils were deteted y stining with 1 lm dihydroethidium (DHE) (Ymmoto et l., 22), nd H 2 O 2

3 S. Tin et l. / Chemosphere 84 (211) ws lolized using with 1 lm roxy-h 2 DCFDA (Moleulr Proes, Eugene, OR) for 15 min nd then rinsed (Freemn et l., 24). The segments were wshed twie in the sme uffer for 15 min eh nd were then emedded in 3% polyrylmide loks (Rodriguez-Serrno et l., 26). A Nikon Elipse 3 epifluoresent mirosope (Melville, NY) equipped with green fluoresent protein filter (exittion nm, emission 5 53 nm) ws used for epifluoresene imges. Exposure times were equl for ll smples. Autofluoresene ws not oserved in unstined ontrols t the exposure time used. Imges were ptured with SPOT mer (Nikon) Determintion of non-protein thiols nd glutthione Non-protein thiols (NPT) were extrted y homogenizing pproximtely thirty root tips (weighed) in 2 ml ie-old 5% (w/ v) sulfosliyli id solution. After entrifugtion t 1 g t 4 C for 3 min, the superntnt ws olleted nd immeditely ssyed. NPT ws mesured with Ellmn s regent (Devos et l., 1992). Briefly, 3 ll of the superntnt ws dded to 1.2 ml.1 M K-phosphte uffer (ph 7.6). After otining stle sorne t 412 nm, 25 ll of 5,5 -dithiois-2-nitroenzoi id (DTNB) solution (6 mm DTNB dissolved in 5 mm EDTA nd.1 M phosphte uffer solution (ph 7.6) ws dded nd the inrese in sorne t 412 nm ws red. GSH ws ssyed y the GSSGreyling method ording to Devos et l. (1992) Antioxidnt enzymes ssys Antioxidnt enzymes were determined ording to the methods desried y Jin et l. (28). Root smples of known weight (1 g fresh weight) were homogenized in 6 ml pre-ooled 5 mm potssium phosphte uffer (ph 7.) ontining.2 mm EDTA nd 2% (w/v) polyvinylpyrrolidone (PVP) in n ie th using prehilled mortr nd pestle. The homogente ws entrifuged for 2 min t 12 g t 4 C nd the superntnt otined ws used for enzyme nlysis. An liquot of the extrt ws used to determine the enzymes ontents. The tivities of SOD isoenzymes were determined ording to Xu et l. (21). The sorne ws reorded t 56 nm. One unit of SOD tivity ws defined s the mount of enzyme required to inhiit the redution rte of NBT y 5%. Ctlse (CAT) tivity ws ssyed in retion mixture ontining 25 mm potssium phosphte uffer (ph 7., ontining.1 mm EDTA), 1 mm H 2 O 2, nd the enzyme. The derese in sorne of H 2 O 2 within 1 min t 24 nm (E = 39.4 mm 1 m 1 ) ws reorded. The gluthione peroxidse (G-POD) tivity ws ssyed in retion mixture ontined 25 mm potssium phosphte uffer (ph 7., ontining.1 mm EDTA),.5% guiol, 1 mm H 2 O 2, nd the enzyme tivity ws mesured y the inrese in sorne t 47 nm used y guiol oxidtion (E = 26.6 mm 1 m 1 ) Trnsmission eletron mirosopy Fresh root tips (out 1 3 mm in length) with different tretments were seleted nd fixed in 4% glutrldehyde (v/v) in.2 M sodium phosphte uffer (ph 7.2) for 6 8 h, post-fixed in 1% osmium tetroxide (OsO 4 ) for 1 h nd wshed in.2 M sodium phosphte uffer (ph 7.2) for 1 2 h. Dehydrtion ws rried out in grded ethnol series (5%, 6%, 7%, 8%, 9%, 95%, nd 1%) followed y etone (1%), then smples were infiltrted nd emedded in Spurr s resin. Ultr-thin setions (8 nm) were prepred nd mounted on opper grids nd viewed under trnsmission eletron mirosope (JEOL TEM-12EX, Jpn) t n elerting voltge of 6. kv Sttistil nlysis of dt All dt were sttistilly nlyzed using the SPSS pkge (Version #11.), nlysis of vrine (ANOVA) ws performed on the dt sets, with the men nd SE of eh tretment lulted. 3. Results 3.1. Root growth nd Cd onentrtion in root tips Inhiition of root elongtion is usully the first visul symptom of Cd toxiity in plnts. Tretment of roots with 4 lm Cd for 24 h hd signifintly inhiitory effet on the root growth of S. lfredii (P <.5), Fig. 1A. The ddition of C to the Cd medium signifintly improved root elongtion while the pplition of C seprtely in the ulture medium did not notly ffet root growth (Fig. 1A). Clium ddition to medium signifintly inresed C ontent (P <.5, Fig. 1B) nd redued Cd umultion y 42.2% (P <.5, Fig. 1C) in root tips of S. lfredii. (A) (B) (C) Root elongtion (% of ontrol) C onentrtion ( µg g -1 DW -1 ) µg g -1 DW -1 ) Cd onentrtion ( Control C Cd Cd+C Control C Cd Cd+C n.d. n.d. Control C Cd Cd+C Fig. 1. Cdmium onentrtion (A) nd root elongtion inhiition (B) in roots of S. lfredii. Seedlings growing in nutrient solution were exposed to four tretments (1) ontrol, (2) 6. mm C, (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd (ph 5.8) for 24 h. Inhiition of root elongtion (perentge of ontrol) ws lulted s (B/A) 1, where A ws the root length elongted in the ontrol plnts, nd B ws the root length in treted plnts. All dt show the mens ± SE of three replites. n.d., not deteted.

4 66 S. Tin et l. / Chemosphere 84 (211) Loss of plsm memrne integrity nd lipid peroxidtion The effet of Cd exposure on plsm memrne integrity nd lipid peroxidtion in roots of S. lfredii, ws determined with histohemil stining with Evns lue (Fig. 2A) nd Shiff s regent (Fig. 2B). The roots of S. lfredii treted with Cd lone were hevily stined y oth regents while those treted with supplementl C hd signifintly lighter stining. Quntittive determintion of Evns lue umultion indited tht the loss of plsm memrne integrity in root tips of S. lfredii ws redued signifintly (P <.5) in the presene of C in Cd medium. Clium ddition to the tretment medium resulted in Evns lue umultion 1.89 times tht of the ontrol, s ompred to 3.15 times of ontrol in the sene of C (Fig. 2C). Quntittive determintion of lipid peroxidtion in root tips of the plnts ws mesured s the ontent of MDA, thiorituri id retive metolite. MDA ontent inresed signifintly in root tips of S. lfredii when the plnts were exposed to 4 lm Cd, while the effet of Cd ws signifintly llevited y ddition of C in the medium (Fig. 2D, P <.5) Retive oxygen speies (H 2 O 2 nd O 2 ) Cd-indued oxidtive dmge in plnt ells hs een linked to the enhned prodution of retive oxygen speies (ROS) (Gehev nd Hille, 25; Zhu et l., 25). The onentrtion of oth H 2 O 2 nd O 2 in root tips of S. lfredii treted with Cd inresed signifintly (P <.5), s ompred with the ontrols (Fig. 3). Applition of C seprtely in the ulture solution in the sene of Cd did not notly ffet ROS produtions in roots of S. lfredii however, ddition of C to the Cd medium signifintly redued the prodution of O 2 (P <.5), nd there ws non-signifint redution in H 2 O 2 umultion in root tips of S. lfredii. To visulize H 2 O 2 nd O 2 prodution, roxy-h 2DCFDA (Freemn et l., 24) (Fig. 3C) nd dihydroethidium (DHE) (Ymmoto et l., 22) (Fig. 3D) ws used, respetively. The speifiity of oth proes ws heked y using speifi ROS svengers (Rodriguez- Serrno et l., 26), ASC for H 2 O 2, nd TMP for O 2 (dt not shown). Under exposure of 4 lm Cd, the overprodution of oth H 2 O 2 nd O 2 ws oserved in vivo in root tips y fluoresene imging. However, the prodution of oth H 2 O 2 nd O 2 were prevented y exogenous C to the Cd medium. A slight fluoresene of H 2 O 2, ut no fluoresene of O 2, were oserved in the root tips from the ontrol plnts, oth with or without pplition of exogenous C (Fig. 3C nd D) Antioxidtive enzymes (SOD, CAT, G-POD) nd ntioxidnts (GSH nd NPT) Regultion of ROS levels n e hieved y the ntioxidtive system omposed of enzymti svengers suh s SOD, CAT nd G-POD (Shutzenduel nd Polle, 22). As shown in Tle 1, totl tivities of oth SOD nd CAT inresed signifintly upon the exposure to 4 lmcd(p <.5). However, SOD nd CAT tivities were notly deresed in the roots of S. lfredii treted with Cd when C ws present (P <.5). Anlysis of SOD isoenzymes indited tht ddition of C signifintly deresed Cd-indued FeSOD tivity (P <.5), ut hs no suh effet on either MnSOD or Cu/ZnSOD (Tle 1). Tretment with Cd strongly inhiited tivities of G-POD in the roots of S. lfredii nd enzyme tivities (A) Control C Cd Cd+C Control C Cd Cd+C (B) (C) Evens lue uptke (% of ontrol) MDA ontent (D) ( µmol g -1 FW) Control C Cd Cd+C Control C Cd Cd+C Fig. 2. Cdmium-indued loss of plsm memrne integrity nd lipid peroxidtion in the root tips of S. lfredii. Seedlings growing in nutrient solution were exposed to four tretments (1) ontrol, (2) 6. mm C, (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd (ph 5.8) for 24 h. [A] mirosope imges of Evns lue uptke in roots tips, [B] mirosope imges of lipid peroxidtion in roots tips of S. lfredii; [C] Evns lue uptke (% of ontrol) in roots tips; [D] MDA ontent in roots tips. Br represents 1 mm; All dt show the mens ± SE of three replites.

5 S. Tin et l. / Chemosphere 84 (211) (A) H 2 O 2 ontent (µmol g -1 FW) (B) O 2 - ontent (µmol g -1 FW) Control C Cd Cd+C Control C Cd Cd+C (C) Control C Cd Cd+C Control C Cd Cd+C (D) Fig. 3. Cd-indued retive oxygen speies in the root tips of S. lfredii. Seedlings growing in nutrient solution were exposed to four tretments (1) ontrol, (2) 6. mm C, (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd (ph 5.8) for 24 h. [A] H 2 O 2 ontents in root tips of S. lfredii, nd [B] O 2 ontents in root tips of S. lfredii, dt show the mens ± SE of three replites; [C] nd [D] represent fluoresene imging of H 2 O 2 nd O 2 prodution in the root tips of S. lfredii, respetively. Br represents 1 mm. Tle 1 Response of superoxide dismutse (SOD), tlse (CAT), guiol peroxidse (G- POD), non-protein thiols (NPT), nd glutthione (GSH) in roots tips of S. lfredii. Enzyme Tretments Control +C +Cd +C + Cd Unit mg 1 protein TotlSOD 39.6 ± ± ± ± 4.8 MnSOD 16.5 ± ± ± ± 3.4 FeSOD 7.2 ± ± ± ± 2. Cu/ZnSOD 16. ± ± ± ± 2.4 lmol mg 1 protein CAT.32 ±.3.33 ±.2.46 ±.4.39 ±.2 G-POD 42.8 ± ± ± ± 3.7 lmol g 1 FW NPT.52 ±.7.63 ± ± ±.12 GSH.2 ±.2.22 ±.3.32 ±.5.42 ±.4 Seedlings growing in nutrient solution were exposed to four tretments (1) ontrol, (2) 6. mm C, (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd (ph 5.8) for 24 h. All dt show the mens ± SE of three replites. Different letters in the sme line indite signifine t P <.5. lrgely reovered with the ddition of C to the Cd-stressed roots (Tle 1). Levels of the most ommon non-enzymti ntioxidnts (NPT nd GSH) were mesured in roots of S. lfredii. Tretment of seedlings with 4 lm Cd for 24 h signifintly inresed the umultion of oth NPT nd GSH in the roots of S. lfredii (Tle 1). Applition of exogenous C in the Cd medium signifintly redued the Cd-indued overprodution of NPT though levels were not redued to those in the unstressed ontrol plnts (Tle 1). Interestingly C supplementtion in the Cd tretment inresed the umultion of GSH (Tle 1) Trnsmission eletron mirosopy A visul omprison of representtive ells viewed under trnsmission eletron mirosopy suggests tht Cd uses signifint ultrstruturl dmge to root tip ells. Under ontrol onditions, the root ells of S. lfredii (Fig. 4A) exhiited qulittively riher ytoplsm with undnt orgnelles, smll vuoles, smooth nd ontinuous ell wlls, with lrge nuleus nd nuleolus. Clerly delineted ell wlls, numerous endoplsmi retiulum, mitohondri nd plsmodesmt were visile (Fig. 4A). Visul omprison with ells from Cd treted roots suggested n dverse effet on ell qulity nd ultrstruturl integrity (Fig. 4B). Clium supplementtion onsiderly llevited visul ell dmge (Fig. 4C). At 4 lm Cd, the meristemti ells of roots of S. lfredii exhiited sprse ytoplsm nd orgnelles inluding some swollen mitohondri, generlly lrger nd more undnt vuoles, nd indistint nuleus nd nuleolus (Fig. 4B). Cell wlls ppered less smooth nd hd mny tthments, nd possily dmged memrne systems (Fig. 4B). For the seedlings exposed to 4 lm Cd with ddition of C, root ells hd etter visul integrity inluding rih endoplsmi retiulum, plsm memrne nd ell wlls tht resemled ontrol ells nd ler nuleus nd nuleolus whih were however, smller thn in ontrol plnts (Fig. 4C).

6 68 S. Tin et l. / Chemosphere 84 (211) Fig. 4. Trnsmission eletron mirogrph of root meristemti ells from S. lfredii. Seedlings growing in nutrient solution were exposed to four tretments (1) ontrol, (2) 6. mm C, (3) 4 lm Cd, nd (4) 6. mm C + 4 lm Cd (ph 5.8) for 24 h. Brs = 2 lm. Lels: C, ytoplsm; CW, ell wll; ER, endoplsmi retiulum; M, mitohondri; NU, nuleus; NUE, nuleolus; PD, plsmodesmt; PL, plsm lemm; V, vuole. 4. Disussion In the presene of rhizotoxi levels of minerl toxints, supplementtion of the medium with higher levels of C hs een shown to llevite growth inhiition (Kinride, 1998; Rodriguez-Serrno et l., 29; Wng nd Song, 29). In the present study, we onfirmed tht supplementtion of the medium with higher levels of C llevites growth inhiition nd oxidtive stress of roots of S. lfredii under the exposure of rhizotoxi levels of Cd. Severl mehnisms for the C llevition of minerl toxiities hve een suggested (Kinride, 1998), one of whih is the restortion of toxint-displed C in plnt orgnelles nd ell surfes (Kinride, 1998; Wng nd Song, 29). It hs een reported tht Cd pplition resulted in derese of C ontent in vrious plnt speies (Gussrsson et l., 1996; Sndlio et l., 21; Rodriguez- Serrno et l., 29; Wng nd Song, 29). The results of the present investigtion onfirm this oservtion s exposure to 4 lm Cd signifintly redued C ontent in the root tips of S. lfredii, s ompred with the ontrols, nd the pplition of C in the solution signifintly reversed this effet (Fig. 1B). In the present study, C pplition redued the Cd ontent in the roots of S. lfredii seedlings (Fig. 1C). Similr meliortive effets of exogenous C on Cd umultion in plnts hs een reported in Trifolium repens L. seedlings (Wng nd Song, 29). These results suggest tht C ompetes with Cd for uptke through C trnsporters (Lu et l., 28, 21). Clium nd Cd likely ompete not only for the root plsm memrne trnsporters ut lso for intrellulr inding sties within plnts nd in C dependent metoli funtion (Rodriguez-Serrno et l., 29; Lu et l., 21). Clium is involved in the regultion of plnt ell metolism nd signl trnsdution nd modultes ellulr proesses y inding proteins suh s lmodulin (CM), whih in turn regultes the tivity of trget proteins (Rodriguez-Serrno et l., 29). In this study, we provide evidene tht C supplementtion is le to redue Cd-indued oxidtive stress in roots of S. lfredii nd to redue lipid peroxidtion nd loss of plsm memrne integrity (Fig. 2), prevent ROS (H 2 O 2 nd O 2 ) over prodution (Fig. 3), nd mintin ultrstruturl integrity of root ells (Fig. 4) in the presene of otherwise toxi levels of Cd. Plnts hve well-developed defense systems to mintin metolilly omptile levels of H 2 O 2 nd O 2 (Alsher et l., 22). The enzymes SOD nd CAT re involved in the detoxifition of O 2 nd H 2O 2, respetively. Within ell, SOD onstitutes the first line of defense ginst O 2 y rpidly onverting it to O 2 nd H 2O 2 (Alsher et l., 22). It hs een suggested tht externl C ould

7 S. Tin et l. / Chemosphere 84 (211) llevite oxidtive stress y improving ntioxidnt enzyme (SOD nd CAT) tivity in Cd-stressed plnts (Rodriguez-Serrno et l., 29; Wng nd Song, 29). In this study, however, redued tivity of SOD nd CAT ws deteted in the C-treted Cd hllenged roots (Tle 1), suggesting tht C depressed genertion of ROS tht trigger the SOD, most likely FeSOD, nd CAT tivity. This suggests tht some other mehnism my ount for the C llevition of ROS overprodution in S. lfredii. GSH is well hrterized ntioxidnt tht plys prominent role in defense system of plnts (Clemens, 26). In these experiments we oserved n inrese in GSH onentrtions in the Cd treted roots nd oserved further stimultion of GSH prodution with the pplition of supplementl C (Tle 1), suggesting tht C llevition for ntioxidtive stress of Cd is proly relted to GSH iosynthesis. A stimultion of GSH iosynthesis with Cd exposure hs een oserved previously in this speies (Sun et l., 27; Jin et l., 28). The ext mehnism of Cd-indued GSH iosynthesis nd C llevition of Cd toxiity in the hyperumultor S. lfredii needs to e further investigted. 5. Conlusion The results of this study demonstrte tht exogenous C llevited Cd-indued growth inhiition nd deresed Cd onentrtion nd inresed C onentrtion in roots of S. lfredii. Lipid peroxidtion nd loss of plsm memrne integrity, ROS (H 2 O 2 nd O 2 ) over prodution, s well s ultrstruturl hnges in root ells indued y Cd were lrgely reversed y C supplementtion. Clium pplition onsiderly llevited the Cd effets on ntioxidnt enzymes nd NPT, ut signifintly inresed GSH iosynthesis. These results suggest tht C protets the roots of S. lfredii ginst Cd toxiity y restortion of Cd-displed C, llevition of the metl indued oxidtive stress, s well s promoted GSH iosynthesis. Aknowledgements This work ws supported y Projet from the Ntionl Nturl Siene Foundtion of Chin (31935), Key Projet from Ministry of Environmentl Protetion of Chin ( ), 863 Trget Gol Projet from Ministry of Siene of Chin (29AA6Z316). Chin Postdotorl Siene Foundtion funded projet ( ). Referenes Alsher, R.G., Erturk, N., Heth, L.S., 22. Role of superoxide dismutses (SODs) in ontrolling oxidtive stress in plnts. J. Exp. Bot. 53, Bker, A.J.M., MGrth, S.P., Reeves, R.D., Smith, J.A.C., 2. Metl hyperumultor plnts: review of the eology nd physiology of iologil resoure for phytoremedition of metl-polluted soils. Phytoremedition Contmin. Soil Wter 85, 17. Bertin, G., Averek, D., 26. Cdmium: ellulr effets, modifitions of iomoleules, modultion of DNA repir nd genotoxi onsequenes ( review). Biohimie 88, Clemens, S., 26. Toxi metl umultion, responses to exposure nd mehnisms of tolerne in plnts. 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